The Herbal Treatment of Depression

Depression is a disorder marked particularly by sadness, inactivity, difficulty with thinking and concentration. A noteworthy increase or decrease in appetite and time spent sleeping, feelings of dejection and desperateness. And sometimes very serious suicidal thoughts or an attempt to commit suicide.

Depression is a state of mind familiar to almost everyone in one form or another. In ordinary usage, the word mentions to a mood state that in medicine is called dysthymia. In contrast with the normal state of euthymia and the opposite state, elation.

In psychiatric usage, disorders of mood are called affective disorders. Depression is either a disorder or can be a symptom of another disorder, either mental or physical. Normal human responses to some situations may include transient depression. But in medicine, these transient states are distinguished from the condition known as clinical depression.

Major depression occurs in 10% to 20% of the world's population in the course of a lifetime. Women are more often affected than men are, by a 2:1 ratio, and they seem to be at particular risk just before menstruation or immediately after childbirth. Relatives of people with major depression also seem to be at higher risk of developing depression, and about 2% of the population may have a chronic the disorder is known as depressive personality.

The Herbal Alternatives to Prozac." most of the talk challenging the assumptions underlying the title! By phrasing the topic in this way, we are forced to respond to the underlying issue in terms of the efficacy of Prozac for depression, rather than by highlighting the strengths of phototherapy when used in a holistic context.

In short, comparing the efficacy of herbs and Prozac begs the fundamental question about the underlying cause of depression. When it comes to the clinical use of herbs in major depressive illness, to make it clear that she does not believe there is an herbal alternative. For most people with less severe depression, however, herbal alternatives will be discussed below.

That would also suggest that depression may be a rational and sane response to our society's initial manifestation of 21st-century culture! As such, is a chemical "smiley face," whether synthetic or botanical, an ethical response? We don't suppress the inflammatory response simply because it occurs, so why suppress the emotions that accompany depression?

Depression can represent an opportunity for self-exploration for the patient, and many lessons maybe learned. Of course, this will entail a great challenge for both the patient and the practitioner. Depression is defined by a standard set of symptoms described in the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders (DSM):

• A mediocre appetite and noteworthy weight loss, or improved appetite and significant weight gain

• Insomnia or increased sleep

• Agitation or retardation of movement and thought

• Loss of interest or pleasure in usual activities, and decrease in sexual drive

• Loss of energy and fatigue

• Self-reproach and feelings of worthlessness, or excessive or inappropriate guilt

• Decrease inability to think or concentrate; indecisiveness

• Negative thoughts of death or suicide, or suicide attempts

Not all these characteristics will occur in everyone who becomes depressed. For determinations of psychiatric treatment, a person is considered to have experienced a foremost depressive episode. If he or she shows a loss of interest or pleasure in all or almost all normal activities. That shows at least four of the above symptoms almost every day for a period of at least two weeks.

The term depression is frequently modified by words that simply either some precise factor or some the chemical mechanism as the cause of the state. For example, depression that is considered a reaction to some loss of or separation from a valued person or thing is called reactive or exogenous depression.

In contrast, the usually, a more severe form of depression without apparent cause is called endogenous depression. Melancholia, a term once used to designate all depressive states is now applied only to these most difficult forms of depression.

Treatment of Depression

In terms of the herbal component of treatment protocols for depression, the nervous system must be the focus for toning. But associated symptoms may be a clue that more deep-seated issues have begun to manifest. Attention to the liver and the digestive system, in general, is usually a good idea.

Actions Indicated

Nervine tonics are fundamental to any long-term change in the individual's ability to cope and transform what must be changed.

Nervine relaxants may be indicated in the short term, or if the depression has an agitated or hyperactive aspect. These should not be strong herbs, which could trigger a more entrenched depression.

Nervine stimulants might help, but not predictably. If the therapist concludes that stimulation is appropriate, it is better to use bitter metabolic stimulants.

Bitters often bring about dramatic changes in patients' perceptions of themselves and of their lives. This highlights the need for a holistic perspective in such conditions.

Antispasmodics will alleviate muscular tension that might manifest as a bodily expression of psychological depression. Care should be taken not to use strong relaxants.

Adaptogens support the adrenals in coping with the stress that the whole body is experiencing.

Hepatics are indicated to support the liver's detoxification work, especially if the patient has been using prescription psychotropic drugs.

Specific Remedies

As far as this concerned, there are no clear-cut specifics for depression. Hypericum perforatum. It has a long tradition of use in Europe, and while it sometimes achieves remarkable results, it also sometimes does nothing. This herb requires time to work, and so must be taken for at least a month.

Of numerous clinical trials performed to investigate the antidepressant effects of St. John's wort extract, a double-blind clinical trial conducted in 1993 concluded that 67% of patients experienced a positive improvement after four weeks of therapy. The study involved 105 outpatients with depressions of short duration who received either 300 mg of St. John's wort extract three times a day or placebo three times a day.

A Prescription for Moderate Depression

·         Hypericum perforatum 2 parts

·         Avena sativa 1 part

·         Artemisia vulgaris 1 part

Dosage: up to 5 ml of tincture three times a day for at least 1-month.

Actions Supplied by Prescription for Depression

·         Nervine tonic - Hypericum perforatum, Avena sativa

·         Nervine relaxant Hypericum perforatum, Avena sativa, Artemisia vulgaris

·         Bitter Artemisia vulgaris

Broader Context of Treatment

The whole gamut of issues touched upon from green salads to relaxation, spinal adjustments to changing the music one listens to—the list of factors to consider is endless. Exercise is especially important.

In Textbook of Natural Medicine, Drs. Pizzorno and Murray suggest the following nutritional supplements:4 B vitamin complex: 50 times the recommended daily dose each day Vitamin C: 1 g three times daily Folic acid: 400 mg/day Vitamin Bu: 250 meg/day Magnesium: 500 mg/day.

Herbal Treatment of Anorexia Nervosa

A disorder characterized by a disturbed sense of body image and marked anxiety about weight gain. And manifested by abnormal patterns of handling food, marked weight loss, and amenorrhea in women. Anorexia nervosa is a problem typified by self-starvation. It occurs most commonly among young women but is also observed in older women and men.

For unknown reasons, anorexia nervosa has become more common in recent years. And the incidence among young women in the United States may be as high as 1% to 2% (0.1% to 0.2% in males). The disorder may appear when the individual first leaves home or may develop in connection with mental depression, peer pressure to lose weight, sexual temptation, or the discontinuation of oral contraceptives.

At the outset, the patient either stops menstruating or simply refuses to eat. In the first case, the patient later stops eating; in the second, her menstrual periods eventually cease. In either case, she may lose weight to the point of life-threatening exhaustion.

In general, the patient will sleep poorly but, despite weight loss, will remain physically active, believing herself to be much fatter than she is. Body temperature may have to be very low. These symptoms suggest that anorexia nervosa may be associated with a disorder of the hypothalamus, a region of the brain that regulates menstruation, eating, body temperature, and sleep.

Herbal Treatment of Anorexia Nervosa

The herbal treatment must focus on toning the nervous system. But in addition, the digestive and reproductive systems will need aid. Bitters and hepatics are especially useful in supporting the liver and digestion.

Actions Indicated

Bitters are indicated because they stimulate both appetite and general metabolism. Nervine tonics are fundamental to any long-term change in the individual's ability to cope with life and transform what must be changed.

Nervine relaxants will alleviate associated anxiety. Hepatics will support the detoxification process and generally benefit the body. System tonics must be used to support any part of the body weakened by the condition or history of illness. In women, anorexia nervosa places the reproductive system under much strain.

Digestive support will be indicated if abdominal symptoms exist. Please refer to the sections on indigestion and irritable bowel syndrome. Immune support may be necessary.  

Specific Remedies

Bitters are considered specifics here, but especially Verbena officinalis (vervain), a relaxing nervine with marked hepatic properties. Because it is strictly a problem

of modern culture. The herbal traditions provide no information about the treatment of anorexia nervosa.

A Prescription for Anorexia Nervosa

·         Gentiana lutea 1 part

·         Avena sativa 1 part

·         Hypericum perforatum 1 part

·         Verbena officinalis 1 part

Dosage: 5 ml of tincture 10 to 15 minutes before eating, three times a day.

Actions Supplied by Prescription for Anorexia Nervosa

·         Nervine tonic - Avena sativa, Hypericum perforatum, Verbena officinalis

·         Nervine relaxant - Hypericum perforatum, Verbena officinalis

·         Bitter - Gentiana lutea

·         Hepatic - Gentiana lutea, Verbena officinalis

Emmenagogues might be indicated if the menstrual cycle has been disrupted. If you feel sleep difficulties, please look to the treatment of Insomnia.

Broader Context of Treatment

As with depression, the whole gamut of issues must be addressed. As with depression, herbal therapy (or drug therapy) does not replace the fundamental need for competent and appropriate psychotherapy.

Laryngitis Treatment

What is Laryngitis?

Laryngitis is an acute inflammation of the larynx, or voice box. It is normally associated with a common cold or overuse of your voice. It is characterized by swelling, hoarseness, pain, dryness in the throat, coughing, and inability to speak above a whisper, if at all.

It is usually caused by a bacterial or viral infection, either restricted to the larynx or part of a more general infection of the upper respiratory tract. Where no clear-cut cause is found, such as infection or overuse, skilled diagnosis is required.

Treatment of Laryngitis

Hence, How to Treat Laryngitis? The lymphatic system should be the focus of tonic support. But if there is a pattern of recurrent infection, attention must also be given to strengthening the immune system.

Actions Indicated

• Demulcents will soothe the mucous lining and ease discomfort.
• Anti-inflammatories will reduce the immediate cause of distress.
• Antimicrobials are indicated if there is a causal microorganism involved. However, they are not indicated if inflammation is due to some other cause.
• Astringents are often effective as a local gargle, especially if the problem was precipitated by overuse of the vocal cords.
• Bitters have a toning and stimulating effect on the mucosal lining.

Specific Remedies

The various herbal traditions of the world abound with herbs effective for conditions of the mouth, larynx, and pharynx. Osha (Ligusticum porteri) is an excellent specific. A small piece of the root can be chewed to ameliorate symptoms and promote the body's immune response.

In Europe, gargling with astringent herbs is the traditional approach. They should not be drunk, as they will probably also promote constipation—an unnecessary and unfortunate complication.

Astringent Herbs Relevant to Laryngitis

• Achillea millefolium (yarrow)
• Geranium, maculatum (cranesbill)
• Polygonum bistortum (bistort root)
• Potentilla to'rmentosa (tormentil)
• Quercus spp. (oak bark)
• Rubus idaeus (raspberry leaf)
• R. villosus (blackberry leaf)
• Salvia officinalis var. rubia (red sage)
• Sambucus nigra (black elder flower)

A Prescription for Laryngitis

• Echinacea spp. 2 parts
• Ligusticum porteri 2 parts
• Hydrastis canadensis 1 part

Dosage: up to 1 ml of tincture every hour

A Gargle for Laryngitis

• Salvia officinalis var. rubia 1 part
• Matricaria recutita 1 part

Make a strong infusion with dried herbs. Gargle often until symptoms subside.

Gargle II for Laryngitis

• Malva neglecta or Malva sylvestris

Infuse Malva flowers and leaves in lukewarm water overnight to ensure optimum extraction of mucilage. Gargle as needed.

Self Care Methods:

• You need to avoid whispering
• You need to avoid decongestants
• You need to moisten your throat
• You need to take plenty of water or fluids to prevent dehydration
• You need to give rest your voice
• You need to breathe moist air

The Purpose of Skeleton in Human Body

This is very interesting thoughts of why do we have a skeleton? There are two main jobs that the skeleton does it supports the body, and it protects delicate organs. The skeleton is the frame that holds man erect. It is made mostly of bones.

A baby is born with as many as 270 small, rather sot bones in his framework. A fully grown person usually has 206, because some bones become fused, or grow together. Bones it together at joints and are held fast by ligaments, which are like tough cords or straps. Some joints can be moved freely. For example, when you run, you move your legs at the hip and knee joints.

When you throw a ball, you move your arm at the shoulder and elbow joints. Moreover, some joints cannot be moved at all. At the base of the spine the bones are fused, forming one bony plate that fits into another.

Neither move, the joints in your skull are very solid, too, except for those in the jaws. The protection that the skeleton provides includes the hard, bony cap o the skull. This protects the brain. The rib cage protects the heart and lungs. And the backbone, or hollow spinal column, protects the spinal cord, the body trunk line of nerve cables. The backbone is actually a string of small bones.

It is very hard for us to think of bone as living tissue, but it is. It grows when a person is young. For example the thigh bone may triple in length between the time a person is born and the time he is fully grown.

Bones grows in length and thickness as calcium and other minerals are added to them. And since bone is living tissue, it must be fed. The outside of the bone is covered with a thin, tough skin. The skin holds many tiny blood vessels that carry food to the bone cells.

The middle of a bone is spongy and filled with marrow. Some of the marrow is a storehouse for fat, and other marrow makes red blood cells. There are six major skeleton purposes, movement, and protection, storage of minerals, endocrine regulation and production of blood cells. Also, men and women have an average 12 pairs of ribs, in some special cases; few have 13 or 11 pairs of ribs.

A human bone grows and changes over the whole life. Furthermore, a healthy skeleton needs daily exercise, walking, jumping, skipping; support the human bones to grow strong. Also, a healthy bone mass in the skeleton reaches utmost density around the age of thirty. Our lungs can collapse without rib cage, and intercostals muscle. 

The Natural Tooth Whitening

Now that you’ve seen the cost of both over the counter and professional teeth whitening procedures! That is get ready to see the wonderful comparison for the price of the natural products you can use to whiten your teeth. For Natural Tooth Whitening prepare yourself - this may shock you after seeing some of the prices. ‘Inexpensive’ doesn’t start to describe the low cost of natural products you can use to whiten your teeth at home.

·         Baking soda, lemon, orange, strawberries- typically less than $1each

·         Apple cider vinegar is usually a couple of dollars for a gallon! Imagine how long a gallon would last when you use it to whiten your teeth.

·         Ash or charcoal - virtually free!

·         Extra virgin olive oil can be found in a grocery store for just $3-$5 dollars per bottle.

·         Salt, you probably already have at home but if not, a container is normally about $1.

So for less than what you’d spend on professional or over the counter treatments to whiten your teeth. You could try all of these natural treatments to see what whitens your teeth the best. Being able to try different methods of whitening your teeth means that you can determine which treatments are best and meeting your whitening needs.

Some stains and discolorations respond better to certain items than others. This way you can find which items work best for your stains and discoloration. Hence, that you can achieve the best whitening effects. And you should continue to use the most effective treatment to maintain your bright, white smile.

Moreover, the repeat applications and touch up whitening can be done as needed at home, with natural products. But without the worry of the side effects that professional bleaching can have. Some side effects of bleaching include:

·         Sensitivity of the teeth to hot and cold once the tooth is damaged to the point where the nerves are extra sensitive to heat and cold you’re stuck with the results. You will then have to eat and drink very carefully for the rest of your life to avoid the shooting pain of sensitive teeth.

·         Gum irritation can be caused by the bleaching solution and can even leave “burns” or sores. Who wants their beautifying treatment to leave sores on their gums or lips?

·         Headache is a common side effect of bleaching treatments. Chemical bleaching is introducing harsh chemicals to your body through breathing, swallowing and absorbing them into your body. Headaches aren’t what you want after you’ve just spent a small fortune to look great. You want to feel like showing off that bright white smile!

·         Tingling, numbness and irritation of the tissues in your mouth! Sometimes these side effects last for several days after a professional tooth bleaching treatment. ·

·         Over-bleaching can cause blue teeth. The bleaching process takes your teeth beyond a nice healthy white to a thin and bluish look. Learn when enough is enough.

·         Bleaching treatments can also cause a sore throat.

When you look at the cost comparison as well as the side effects it is easy to see that natural tooth whitening is easier on your mouth and your wallet! Some people will want to consult a dentist to get a treatment program cost and schedule before deciding to try natural tooth whitening at home.

Therefore, this is a good idea because it will allow you to see what you would have to spend. Also how many visits you would need based on the staining and discoloration of your teeth. And the schedule to maintain your newly white teeth once you achieve the desired effect!

Then, when you do it at home with natural products you will know how much money, hassle and effort you’ve saved. Again no one can make the decision for you. But it’s important to know that you do have options that cost less and are less chemically harmful than professional whitening treatments.

The best option for Natural Tooth Whitening for you is the one that works for your schedule, your budget and your teeth whitening needs. No matter whether you decide to use a natural tooth whitening plan or visit a professional for a bleaching treatment.

For removing stains and discoloration from your teeth is an important part of improving your smile. There’s no need to hide your teeth behind closed lips, go ahead and smile big, your newly whitened teeth will sparkle and shine gleaming white.

How Do Our Teeth Grow?

How Do Our Teeth Grow? Healthy teeth are most prominent part of your personality. That helps us to smile at any place, take bite, can speak, or even have your favorite food without any problem. Everybody tooth has the same two parts; a root, or roots; to anchor it in the jaw bone, and a crown. That part can be seen in the mouth.

There are four different materials in a tooth. The “enamel” is hard and shiny and covers the crown. The “”cementum” is a bone like material that covers the root. The “dentine” is an ivory like material that forms the body of the tooth. The dental pulp is made up of tissue that contains nerves, arteries, and veins. These are entering the tooth through an opening at or near the root end.

Lack of calcium or vitamin D in the diet will result in poor enamel which will encourage early decay. The process of dental decay is also aggravated by the collection of sugary or starchy foods around the teeth, especially during the night.

As they decompose in the mouth, these foods produce acids that will act on the calcium of the teeth and make them soluble, causing the teeth to soften, and thus allow bacteria to attack them. Moreover, why do teeth sometimes grow “crooked”? The reason varies with each person, but scientists say that the way the jaws have developed in modern man can cause this problem.

It seems that man’s jaws today do not always provide enough room for his teeth. So, they appear in a crooked position or become shifted during the period of growth. If this happens to a tooth in a lower aw, the opposing tooth in the upper jaw also becomes crooked in position.

This is sometimes causes teeth to stick out, or pushes the lips out, or makes the chin recede, and spoils the appearance of the mouth. The tooth development process is normally separated into five stages:

1. The initiation stage,

2. The bud stage,

3. The cap stage,

4. The bell stage,

5. The maturation stage,

However, the tooth development stage is very difficult to decide at which stage should be assigned to a particular developing tooth. Also, an average person hold only 28 teeth, but it is really painful when thirty-two teeth try to fit in a mouth.

Hence, the extra four teeth are called third molars and also famous as wisdom teeth. At the age of 13, all 28th teeth fit in their place. By the age of 21, a person has 16 permanent teeth at upper jaw and 16 teeth at lower jaw. So it is very important to understand How Do Our Teeth Grow?

Garlic - Food and Medicine

The use of garlic is supported by both an ancient history and a wealth of modern research. More than 3,000 scientific papers cover its chemistry, pharmacology, and clinical uses. The therapeutic uses of garlic are extensive. But those specific to the cardiovascular system include reducing elevated cholesterol, preventing atherosclerosis and hypertension, treating poor circulation to the legs, and improving overall blood flow through antiplatelet actions.

One study involved two groups of subjects: one of 20 healthy volunteers and the other of 62 patients with coronary heart disease and elevated serum cholesterol. Both study groups ate garlic for six months. All involved experienced beneficial changes, which reached a peak at the end of eight months.

The improvement in cholesterol levels persisted throughout the two months of clinical follow-up. The clinicians concluded that garlic essential oil possesses a distinct hypolipidemic (fat-reducing) action in both healthy people and patients with coronary heart disease.

Great attention is being given by research workers to the value of such findings in humans. Several clinical comparisons of the influence of garlic have been published. In one example, a group of volunteers consumed a high-fat diet for seven days.

On day eight of the study, fasting blood was analyzed for the content of cholesterol and other fats. The subjects then received a high-fat a diet supplemented with garlic for seven days; on day fifteen, fasting blood was analyzed again. Results showed that the fat-rich diet significantly increased cholesterol levels, compared with a normal diet.

However, adding garlic to the high-fat diet significantly reduced serum cholesterol levels.15 Based on research investigating the effect of raw garlic on normal blood cholesterol levels in men, Indian research scientists advocated its daily use as a means of lowering blood cholesterol.

Garlic also possesses antiplatelet effects, or an ability to inhibit unnecessary clotting within the blood vessels. It appears to work by reducing the "stickiness" of blood platelets, decreasing platelet aggregation and inhibiting the release of clotting factors in the blood. The garlic constituent thought to be responsible for

this effect is allicin, unique thiosulfate well known for its strong antibiotic and antifungal properties.

An exciting finding indicates that garlic works selectively, inhibiting the synthesis of enzymes involved in plaque formation while sparing the vascular synthesis of important prostaglandins. This finding suggests that garlic would make a safe and effective antithrombotic agent. The traditional use of both garlic and onion in the treatment of hypertension is supported by research.

A study revealed that onion oil contains a blood pressure-lowering prostaglandin. Interestingly, while garlic's antimicrobial effects are lost during cooking, its blood pressure-normalizing and cholesterol-lowering actions appear to be unaffected.

What is Malignant Pleural Mesothelioma?

Malignant pleural mesothelioma is a rare but fatal tumor caused mainly by asbestos exposure. There is no standard treatment as mesothelioma is primarily resistant to all treatments including chemotherapy. Asbestos-induced oxidative stress is thought to play an essential role in the pathogenesis of mesothelioma in the process possibly increasing the expression of the major antioxidant defense mechanisms of the cells.

Both chemo and radiotherapy act at least partly by provoking reactive oxygen species (ROS) generation suggesting a role for the intracellular antioxidants in drug resistance. Other mechanisms associated with drug resistance include the plasma membrane drug transporters, of which several are also redox-regulated.

In the expression and possible role of the major antioxidant enzymes (AOEs) the manganese super-oxide dismutase (MnSOD), catalase, and mechanisms closely related to glutathione (GSH) metabolism was investigated in the biopsies of malignant mesothelioma and/or cell lines in culture.

The methods included Northern Blotting, Western Blotting analysis, immunohistochemistry and measurement of specific enzyme activities. Cell damage after oxidant or cytotoxic drug exposures were analyzed by lactate dehydrogenase release, depletion of high-energy nucleotides and microculture tetrazolium dye assay. MnSOD was highly expressed in mesothelioma tumor biopsies in vivo and cell lines in vitro-compared to non-malignant mesothelial cells.

Mesothelioma cell line expressing the highest MnSOD (10 fold compared to non-malignant mesothelial cells) levels also had the highest levels of GSH, glutathione S-transferase (GST) and catalase, and was the most resistant cell line to oxidants and cytotoxic drugs.

In contrast to mesothelioma cells, lung A549 adenocarcinoma cells, that represent an oxidant and drug-resistant cell line, contained similar levels of MnSOD as non-malignant mesothelial cells. They, however, also contained higher intracellular GSH levels and catalase than mesothelioma cells and had elevated levels of γ-glutamylcysteine synthetase (γGCS). The rate-limiting enzyme in GSH biosynthesis. in contrast to tumor necrosis factor-α (TNFα), cytotoxic drugs failed to induce MnSOD mRNA, protein or activity in A549 cells.

The endogenous level of MnSOD or its induction by TNFα did not explain the oxidant resistance of these cells. GST could not explain the resistance of adenocarcinoma cells. As the activity of total GST was lower in adenocarcinoma cells than in more sensitive mesothelioma cells?

The role of GSH and catalase were also investigated by treating the mesothelioma cells and A549 adenocarcinoma cells with buthionine sulfoximine (BSO), to block glutathione synthesis, and aminotriazole (ATZ) to inhibit catalase. Both BSO - and ATZ - treatment enhanced H2O2 toxicity in three mesothelioma cell lines. While only the depletion of glutathione increased epirubicin toxicity.

BSO treatment also significantly potentiated cisplatin-induced cytotoxicity in mesothelioma and adenocarcinoma cells. Given the obvious importance of GSH in the oxidant and drug resistance of these tumors, altogether 34 mesothelioma tumor biopsies were investigated for both subunits of γGCS.

The catalytic, heavy subunit of γGCS was highly expressed in most of the cases, whereas the regulatory, light subunit (γGCSl) expression was weaker. No expression of these proteins could be detected from the non-malignant mesothelium. The integral membrane drug transporter, P-glycoprotein (P-gp), immunopositivity was found in 61 %, and multidrug resistance proteins 1 and 2 (MRP1 and MRP2) immune positivity in 58 % and 33 % of 36 mesothelioma biopsies.

Normal mesothelium did not express these multidrug-resistant proteins. There was no significant association between tumor proliferation, apoptosis or patient survival and expression of the multidrug-resistant proteins. In conclusion, the simultaneous induction of multiple antioxidant enzymes can occur in human mesothelioma cells.

In addition to the high MnSOD activity, H2O2-scavenging antioxidant mechanisms, γGCS, GST and GSH can partly explain the high oxidant and drug resistance of these cells in vitro; the role of catalase during heavy oxidant exposure is possible. MnSOD can be induced by TNFα, but the induction, however, does not provide any protection against repeated oxidant exposures.

Many mechanisms contributing to the resistance of mesothelioma remain to be investigated, but γGCS may play an important role in the primary drug resistance of this tumor in vivoin maintaining the intracellular glutathione-level. The multidrug resistance proteins P-gp, MRP1, and MRP2 are expressed in mesothelioma cells, but are not likely to be responsible for the primary drug resistance of this malignancy.

Mesothelioma is a tumor derived from the serosal lining of the pleural, peritoneal or pericardial cavities and is most commonly situated in the pleura. Mesotheliomas are rare tumors, accounting for only about 1% of all cancer deaths in the world. Pleural mesothelioma is in approximately 85-90% of cases an asbestos-initiated lethal malignancy. The latency period is about 20 to 40 years.

Accordingly, the peak in mesothelioma cases is expected in 2010, although the asbestos usage in most industrialized countries has been abolished from the 1980s. The prognosis of mesothelioma is poor, as it is highly invasive and primarily resistant to all treatments. Which is including radiotherapy and cytotoxic drugs. A major factor in the pathogenesis has been considered asbestos-induced oxidative stress, which in turn is known to induce several antioxidant mechanisms in the cells.

Mesothelioma provides a vital model for cancer research of a therapy-resistant malignancy in which antioxidant mechanisms may at least partly explain the resistance. Intracellular antioxidants offer protection not only against reactive oxygen species (ROS) but may also modulate the response to different chemotherapeutic drugs that are used in cancer treatment.

Manganese superoxide dismutase (MnSOD) that scavenges superoxide radicals has a controversial role in cancer biology. It has been suggested to be a cancer suppressor. But on the other hand, it offers protection against oxidative stress and thereby may confer resistance against oxidant producing drugs. MnSOD is overexpressed in only some malignant tumors, but its importance in drug resistance is unsolved.

Glutathione has in many studies have been linked with drug resistance both for its role as an antioxidant but also for its function in detoxification reactions. The attention has been drawn to the enzymes in glutathione biosynthesis and how the cell maintains its glutathione level.

Several studies have also been done with other mechanisms that utilize intracellular glutathione and transport it extracellularly. Glutathione S-transferases are a family of detoxification enzymes that are often associated with chemoresistance.

However, the activity of these enzymes is unidentified in mesothelioma. Even though polymorphism of GSTM1 has been linked to the development of this disease. Catalase in addition to glutathione takes part in scavenging excess hydrogen peroxide in the cells. Not many studies link it to drug resistance of malignant cells, but its role should be clarified in oxidant and drug resistance of mesothelioma.

The classical inducers of multidrug resistance are the drug export pumps in the plasma membrane that have different substrate specificities. P-glycoprotein has been studied most, but the recently discovered MRP family offers new avenues for investigators in cancer biology. The first members in the MRP family, MRP1 and MRP2, are dependent on intracellular glutathione and they transport glutathione-conjugated substrates.

In mesothelioma, these mechanisms have not been thoroughly studied before. This series of studies were designed to systematically investigate the expression of the most important antioxidant pathways and drug transporters in mesothelioma cells in vitro and tumor biopsies in vivo.

Besides investigating the expression of these mechanisms, their role in oxidant and chemotherapeutic drug resistance was assessed in vitro. The expression of the AOEs and related proteins was also correlated with tumor growth and patient survival.

History

In 1767 J. Lieutaud recognized two pleural tumors in a series of autopsies, but Wagner was the first to describe the pathology of a primary malignant pleural tumor in 1870. The term mesothelioma was first used by Eastwood and Martin in 1921.

In 1960 Wagner reported 33 cases of diffuse pleural mesothelioma in South Africa, in an area of crocidolite mining. Of these 33 patients, 32 had a history of asbestos exposure and this connected mesothelioma with asbestos. The first reports of mesothelioma in Finland are from the 1960s.

Epidemiology

Mesothelioma is a a rare disease, but its incidence keeps increasing despite the industrial restriction of asbestos usage from the 1980s, as the latency period is approximately 20 -40 years. About 70 cases of mesothelioma are diagnosed in Finland every year.

It has been estimated that the peak of mesothelioma incidence in Finland will be around 2010, with approximately 100 cases per year. The peak incidence has been already achieved in the U.S, but e.g. in Britain, the number of cases per year is climbing and is expected to increase to more than 3000 cases per year.

Mesothelioma is more common among men, only about 10% occur in women. In about 80-90% of the male cases, obvious asbestos exposure is known. In females, it has been suggested that only 23% of mesothelioma cases are asbestos-related. Sporadic cases among children and infants occur.

Etiology

Asbestos is the single most important causative agent of mesothelioma, and the exposure to asbestos fibers is usually occupational. Other lung diseases are caused by asbestos as well, including asbestosis, lung cancer, pleural plaques, pleural fibrosis, pleural effusions, and pseudotumors. Factors determining the risk of mesothelioma include the fiber type, time from exposure, fiber dimensions and fiber surface properties.

There is evidence that persons with greater intensity and duration of asbestos exposure have a higher risk for mesothelioma which, however, can develop with minimal exposure. Therefore, the causative role of asbestos is difficult to rule out as most adults in the industrialized world have asbestos in their lungs. Fibers greater than 8 μm in diameter are most commonly associated with mesothelioma.

Asbestos is a commercial term for a variety of naturally occurring hydrated fibrous silicates. The material is subdivided into two groups, serpentine fibers, and amphiboles. The capacity of different types of asbestos fibers to induce mesothelioma seems to be greatest with amphiboles like amosite (“brown asbestos”) and crocidolite (“blue asbestos”), whereas the serpentine fiber chrysotile (“white asbestos”) is not as tumorigenic.

Chrysotile comprises 90% of the asbestos used worldwide. In Finland, however, the main asbestos used has been anthophyllite, which is one of the amphiboles. It is associated with asbestos-induced diseases such as asbestosis and pleural plaques. Mesothelioma cases are rare, but some have been reported.

Non-asbestos causes of mesothelioma have not been revealed in epidemiological studies, but theoretically, any agent injuring pleura may cause mesothelioma. These include chemical agents, chronic inflammation, viruses, and radiation. Smoking does not increase the risk of mesothelioma. A possible connection to Simian Virus 40 (SV40) was suggested.

In the late 1950s and the early 1960s polio vaccines were contaminated with SV40 and millions of people were exposed. In Finland, vaccines were not contaminated and none of the mesothelioma patients in Finland had received a contaminated vaccine. SV40 large T-antigen has been detected in a high proportion of mesothelioma tissue specimens. However, other SV40-like DNA sequences were also found in non-malignant pleural diseases.

The role of SV40 is unclear, even though in the United States and many other parts of Europe the consensus seems to link it to mesothelioma. Genetic susceptibility is associated at least with some detoxification enzyme polymorphisms. That is including the homozygous deletion of GSTM1 gene or slow acetylation-associated N-acetyl transferase-2 (NAT2) genotype.

Pathogenesis and pathology

The inhaled asbestos fibers must be transported to the pleural cavity to reach the target cells. Parietal pleura are often more extensively involved, but usually, it is difficult to determine if mesotheliomas begin in the visceral or parietal pleura. When inhaled into the respiratory bronchioles and alveoli, chrysotile fibers are usually fragmented by organic acids and cleared by macrophages.

Amphiboles are not as easily decomposed and may remain unchanged for years/decades. The asbestos fibers are transported to the pleural cavity via the lymphatic pathway or by penetrating to the visceral pleura. Amphibole fibers concentrate on certain areas of the parietal pleura, called black spots, that are openings of lymph vessels, and at these spots, the pleura is exposed for years to the effects of asbestos fibers and toxic reactive oxygen species (ROS).

Free radicals and other toxic oxygen metabolites are considered important in the pathogenesis of mesothelioma. Fibers themselves have redox properties as they contain ferrous iron which catalyses the reaction forming ROS.

ROS are also formed indirectly when phagocytic cells meet the fibers; macrophages and neutrophils are known to liberate ROS after asbestos exposure. These active oxygen intermediates can participate in the oncogenic process by many different mechanisms. Genotoxicity, lipid peroxidation, and oncogene modulation are all possible effects of ROS. The long latency period suggests cumulative genetic, cytotoxic and proliferative events.

Pleural mesothelioma is divided histologically into three classes (Travis et al., 1999). The epithelial subtype comprises about 54% of all mesothelioma cases. Epithelial mesotheliomas may be predominantly composed of acinar structures, and differential diagnosis from adenocarcinoma is often demanding.

Other variants of epithelial mesothelioma also exist. Sarcomatotic mesotheliomas, that histologically resemble fibrosarcomas, represent approximately 20% of the cases, and the rest of the cases fall into biphasic mesotheliomas, representing about 25% of the cases.

Clinical features and diagnosis the average age of a patient at the time of diagnosis are approximately 60 years, and there is a strong male predominance. The first symptoms include chest pain, dyspnea, weakness, and cough. Usually, the diagnosis is delayed due to the non-specificity of the symptoms. Thoracic radiograph initially shows pleural effusion in 92% of cases, usually on one side. Only in 7 % of a multinodular pleural tumor without fluid is seen.

In early cases of mesothelioma, nodules or plaques of varying size can be detected in the parietal pleura. Serosal thickening and consequent effusion are often marked. Most cases are unicavitary. Mesothelioma seldom sends metastasis, but it is highly invasive, e.g. to the pericardium.

One of the first diagnostic procedures are cytology of pleural fluid that gives positive results in approximately 30% of cases. Another method used for a diagnostic workup is the computed tomography (CT) scan. The diagnosis is established by biopsy via thoracoscopy in most of the cases. Examination of biopsy of parietal and visceral pleura is the most reliable method for diagnosis.

Histological diagnosis is, however, difficult because of structural variability between different tumors and even within the same tumor, the main problem being differential diagnosis from metastatic adenocarcinoma of the lung. Other differential diagnostic difficulties arise from benign mesothelial hyperplasia and sarcomas in cases of sarcomatoid mesothelioma. In addition to the typical histopathology, panel of immunohistochemical stains will often suggest the right diagnosis.

Many antigens stain positively in adenocarcinoma but remain negative in mesothelioma. The markers used in the diagnostic procedure include the carcinoembryonic antigen (CEA), glycoprotein markers Leu-M1, Ber-EP4 and B72.3, and others like epithelial marker antigen (EMA) and human milk fat globulin-2 (HMFG-2).

In epitheloid tumors, diastase resistant neutral mucin is positive in approximately 70% of adenocarcinomas, but usually negative in epithelial mesothelioma. In the case of sarcomatoid mesothelioma cytokeratins like CK 5/6 and AE1/AE3 are used, as they are generally positive in sarcomas and negative in sarcomatoid mesothelioma.

Calretinin, that reveals the mesothelial origin, is usually positive in mesothelioma and negative in sarcoma and its specificity is over 90%. In differentiating between reactive and neoplastic mesothelium attention should be focused on the degree of cellular atypia and the presence of collagen necrosis that are highly suggestive of malignancy.

Treatment and prognosis

Treatment of malignant mesothelioma remains disappointing, and there is no standard treatment. As in other malignant tumors, surgery, radiation therapy, chemotherapy, supportive therapy or a combination of different modalities are used.

No treatment has so far been shown to offer better survival than supportive therapy alone. Median survival time from diagnosis is less than one year, 5-year survival is less than 5 %. Some factors, however, indicate a more favorable prognosis, including epithelial subtype, age < 65 years, good clinical condition with no weight loss, and absence of visceral pleura involvement.

Surgery alone does not improve survival but may be beneficial for palliation. Four different surgical methods are in use: extrapleural pneumonectomy, pleurectomy/decortication, limited pleurectomy and thoracoscopy with talc pleurodesis.

Extrapleural pneumonectomy is often used in the combination with radiotherapy. Radiotherapy is also used for palliation, especially in cases with pain. Sometimes the disease may regress, but significant improvement in survival has not been achieved.

Radiotherapy is usually given in combination with either surgery or chemotherapy, so the individual effects of the treatment modalities are difficult to document. Many different chemotherapeutic agents have been tested either as a single-agent treatment or in combination therapy.

In the best clinical series, objective responses are seen after single-agent therapy in about 20-30% of patients, but no significant effect on the overall survival. The best results in single-agent treatment have been achieved using anthracyclins, with doxorubicin giving up to a 40% response rate and high-dose cisplatin a response rate of up to 33%.

Rather promising results have been achieved also with carboplatin, epirubicin, ifosfamide, and mitomycin. High-dose methotrexate treatment resulted in a response rate of 37% in a study of 63 patients. Combinations of cisplatin, doxorubicin or an alkylating agent like ifosfamide have been studied, usually two or three drugs are combined.

No clear advantage over single-agent therapy has been observed. Combination therapy with cytokines, like interferon-á, has been disappointing despite the promising results in in vitro studies (Boutin et al., 1998). The resistance mechanisms of mesothelioma tumors have been studied only in few publications and therefore remain largely unknown. Some of these studies will be discussed later.

Lung Cancer

Given the difficulties between the differential diagnosis of mesothelioma and lung cancer, it has included experiments also on the biopsies and cell lines of lung cancer, mainly lung adenocarcinoma. The incidence of lung cancer is increasing due to the habit of tobacco smoking in the world. Over 3 million lung cancer deaths have been estimated worldwide in the year 2000.

In Finland 2 075 new lung cancer patients were diagnosed in 1994, after five years only 10% are still alive. Lung cancer is the second most common cancer among men in Finland.

The incidence among women is climbing and at present lung cancer is the second most common cause of cancer deaths among women. Tobacco is the most important etiological agent of all four subtypes of lung cancer responsible for approximately 90% of all cases.

Other known exogenous risk factors for lung cancer include asbestos, ionizing radiation, and other environmental carcinogens e.g. polycyclic aromatic hydrocarbons, nitrosamines, and aromatic amines.

The endogenous, host-related factors, include immunological factors and genetic predisposition mainly differences in carcinogen metabolism, DNA repair, and altered proto-oncogene and/or tumor suppressor gene expression.

Lung cancer is divided into two major classes mainly for the treatment purposes: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Virtually all cases arise from the epithelial tissue and are bronchogenic carcinoma subtypes. SCLC (30% of all lung cancers) proliferates fast, often sends metastases and is primarily sensitive to anti-cancer drugs.

Therefore, the initial treatment is chemotherapy. However, resistance to treatment develops rapidly and many different resistance mechanisms have been speculated. P-glycoprotein cannot solely explain the clinical drug resistance and other possible drug resistance mechanisms include multidrug resistance proteins and decreased expression of topoisomerase II.

NSCLC comprises three histologically different carcinomas: adenocarcinoma (30-35%), squamous cell carcinoma (30-35%), and large cell anaplastic carcinoma (5%). The treatment of NSCLC is primarily surgery. Combination chemotherapies are widely used for the treatment of NSCLC since only 10-20% of the cases can be operated.

In contrast to SCLC, NSCLC is primarily resistant to single chemotherapeutic agents. In adenocarcinoma, glutathione-related mechanisms have been suggested as potential resistance inducers along with other classical resistance mechanisms.

Reactive oxygen and nitrogen species

A free radical is defined as a chemical species that has a single unpaired electron in the outer orbital. In this state, the radical is extremely reactive and unstable. The most important radicals are the superoxide radical (O2-.), the hydroxyl radical (OH.), nitric oxide (NO.) and peroxynitrite (ONOO-). Reactive oxygen species (ROS) include free radicals and other oxygen-related reactive compounds, such as hydrogen peroxide (H2O2).

ROS are generated in normal aerobic metabolism in mitochondria, which are the main site of production of radicals. In the cytosol and plasma membrane, ROS are formed by NADPH oxidase, cytochrome P450 oxidase and xanthine oxidase. Transitional metals, such as iron and copper, are potential promoters of free radical damage, as they can convert superoxide, which in normal conditions is poorly reactive, into a rapidly reactive and highly toxic hydroxyl radical by Fenton chemistry.

In Haber-Weiss reaction, hydroxyl radical is generated from O2 -. and H2O2. NO. has many useful physiological functions, but in excess amounts is a toxic-free radical as well. Many exogenous agents, such as hyperoxia, radiation, asbestos fibers and ozone induce free radical formation in the cell.

Asbestos fibers cause oxidant production directly and indirectly, one of the ways being catalysis by the ferrous iron, as asbestos fibers have a high iron content. Inflammatory cells, such as neutrophils and alveolar macrophages, also produce large amounts of ROS when activated, especially when the phagocytosis is incomplete.

No production is also activated via the induction of inducible nitric oxide synthase by TNF and other cytokines released from the inflammatory cells. Reactive nitrogen species that are formed in reactions of NO. and oxygen/superoxide mediates the harmful effects of NO.

The pathological effects of ROS are wide-ranging; these toxic products can cause injury practically too all cellular components. Lipid peroxidation of membranes, non-peroxidative mitochondrial damage, lesions in DNA, and cross-linking of proteins are the most relevant reactions of ROS leading to cell injury.

ROS are thought to be especially important in lung tissue that is exposed to much higher concentrations of oxygen than most other tissues, but also to cigarette smoke and environmental pollutants. In addition to the toxic effects, ROS are important in non-toxic cellular reactions, including signal transduction.

Antioxidants

To protect themselves from the harmful effects of oxidants, cells have several antioxidant enzymes and other antioxidant mechanisms. The latter include glutathione (GSH) and numerous GSHdependent enzymes, metal binding proteins, and vitamins.

The three main types of antioxidant enzymes are the superoxide dismutases (SODs), catalase (CAT) and peroxidases, of which glutathione peroxidases (GPx) are thought to be the most important.

The SODs dis-mutate the superoxide radical into H2O2. GPx and CAT reduces H2O2 into water and oxygen. Glutathione redox cycle provides the cell with reduced glutathione (GSH) to act as co-substrate for the peroxidases but to also participate in detoxification reactions and react nonenzymatically with OH and peroxynitrite.

Other enzymes involved in glutathione metabolism are glutathione reductase, glucose-6-phosphate dehydrogenase, glutathione S-transferases and the enzymes participating in GSH-synthesis: -glutamylcysteine synthetase (GCS) and glutathione synthase (GS). Metal-binding proteins ferritin, ceruloplasmin, transferrin, haptoglobin, and albumin contribute to the antioxidant system by inactivating catalytic metals.

The most important antioxidant vitamins include -tocopherol, ascorbate, B-carotene, and flavonoids, but they will not be discussed in this review. Other enzymes with antioxidant capacity include cysteine-containing proteins such as the families of thioredoxin, glutaredoxin, and peroxiredoxin. These may play a role in the resistance of cells against oxidants but also against free radical generating drugs.

Superoxide dismutases

Two main forms of SOD exist intracellularly: a copper-zinc containing superoxide dismutase (CuZnSOD) and a manganese-containing superoxide dismutase (MnSOD). CuZnSOD is found in the cytoplasm and MnSOD in the mitochondria.

Extracellular SOD (ECSOD) is in the extracellular matrix. MnSOD (also known as SOD2) is a homotetramer with a molecular weight of 88 000 and is in the mitochondrial matrix close to the electron transport chain, where ROS are produced in normal cellular metabolism.

The gene is in the long arm of chromosome 6 and is transcribed as two distinct mRNAs of 1 kb and 4 kb. MnSOD is synthesized in the cytoplasm as a precursor molecule containing a leader signal, which is removed during the transport of the molecule to the mitochondria.

Two polymorphic variants of MnSOD have been described, one leading to altered mitochondrial targeting of the enzyme and the other possibly to changed MnSOD in vitro activity. The importance of MnSOD for normal physiology has been proven with knockout mice lacking the MnSOD gene, who died within 10-20 days of neurological manifestations and cardiotoxicity.

Heterozygous mice with half of the MnSOD activity have increased age-related mitochondrial oxidative damage. Approximately 15% of the total intracellular SOD activity is due to MnSOD. In eukaryotic cells, the MnSOD gene regulation is complex. The MnSOD promoter contains binding sites for several transcription factors such as AP1, AP2, SP1, and NF-B.

It has been hypothesized that the oxidative state of the cell is essential in regulating MnSOD expression. MnSOD is induced by the cytokine tumor necrosis factor (TN). TNF binds to its plasma membrane receptor, which initiates a series of events including intracellular ROS production, activation of NF-êB and induction of the MnSOD gene. The TNF induction of MnSOD is blocked by the antioxidant N-acetyl cysteine (NAC).

Other factors that induce MnSOD are hyperoxia, irradiation, oxidized LDL, interleukin-1, interferon-, lipopolysaccharides, H2O2 and asbestos fibers. In some studies, the MnSOD gene induction is associated with resistance to hyperoxia, which would indicate that oxidant stress induces the enzyme to protect from subsequent oxidant injury. However, in contrast to many in vivo hyperoxia models, MnSOD is not directly upregulated by high oxygen tension in human bronchial epithelial cells in vitro.

In human lung, MnSOD is found in type II pneumocytes, bronchial epithelial cells, and alveolar macrophages. High levels of MnSOD are also found from the heart, brain, liver, and kidneys. In human malignancies, the role of MnSOD is controversial. In carcinogenesis, the antioxidant – oxidant imbalance is considered significant.

A polymorphism of the MnSOD gene resulting in an alteration in the transport of MnSOD into the mitochondria due to conformational change in the protein is a risk factor at least for the development of breast and lung cancers. Most studies have shown that MnSOD activity is low in cancer cells, and it has been proposed to be a cancer suppressor gene.

Transfection studies, in which only the MnSOD gene has been introduced, have shown a decreased level of malignancy and transformation of the malignant phenotype to the direction of a non-malignant one. However, interpretation of this study is problematic as transfection creates imbalanced conditions in the cell.

On the other hand, at least gliomas, thyroid carcinomas, esophageal carcinomas, and colon carcinomas appear to contain high MnSOD levels when compared to the non-malignant tissues. In a study of five samples of lung tumors, the activity of total SOD was somewhat lower than in normal lung tissue. In mesothelioma, MnSOD has not been previously studied before our group reported elevated activity of MnSOD in mesothelioma cell lines.

It has been reported that MnSOD is not inducible in cancer cells as it is in non-malignant cells (Wong & Goeddel, 1988) but also this issue is controversial. At least human lung adenocarcinoma cells show MnSOD induction by TNF. Human A549 lung cells also represent a malignant cell type but appear to show MnSOD induction.

CuZnSOD (SOD1) is a homodimer with a molecular weight of 32000 and is localized mainly in the cytosol, but it is also found in the nucleus and peroxisomes. The gene is in chomosome 21, the gene is transcribed as two mRNAs, 0.9 and 0.7 kb, respectively, the latter being the predominant form. In contrast to MnSOD, CuZnSOD-deficient animals and cells are viable, but they are sensitive to oxygen toxicity. Mutation of this gene is associated with familial amyotrophic lateral sclerosis.

The regulation of CuZnSOD also differs from MnSOD, e.g. its level is constitutive in several animal studies and human lungs, and neither is it induced by hyperoxia, TNF or interleukin-6. In a healthy human lung, CuZnSOD is found from the bronchial epithelium.

High levels are also found from the liver, erythrocytes, brain, and neurons. In a recent study, CuZnSOD gene was found to be upregulated in a mesothelioma cell line compared to a non-malignant mesothelial cell line when assessed in a microarray containing over 6900 genes.

Otherwise, its expression and role in human tumors remain unclear. ECSOD is a copper and zinc-containing homotetrameric glycoprotein. It is in the extracellular matrix in all human tissues and its gene is in chromosome 4. ECSOD is induced by cytokines like TNF, direct oxidant stress does not affect ECSOD like it does MnSOD.

In a healthy lung, ECSOD is concentrated in pulmonary vessels and airways and found from systemic arteries. Of the pulmonary cell types, it is found from bronchial epithelium, alveolar macrophages, and endothelial cells. Its role and regulation in cancer are unknown.

Glutathione

Glutathione is the predominant intracellular low molecular weight thiol in all mammalian cells. It is usually present in the mill molar range; the intracellular level is approximately 1-8 mm and the extracellular level typically 5-50 M. About 99% of the intracellular glutathione is in the reduced form.

Approximately 85% of the intracellular glutathione is in the cytosol, about 15% in the mitochondria and a small percentage in the endoplasmic reticulum. The mitochondrial GSH pool is maintained by the activity of a mitochondrial transporter that translocates cytosolic GSH into mitochondria.

GSH is a central protective antioxidant against free radicals and other oxidants, but it has also an essential role in detoxification reactions. Other cellular events where glutathione is considered valuable is modulation of redox-regulated signal transduction, regulation of cell proliferation, remodeling of extracellular matrix, apoptosis, mitochondrial respiration and a reservoir of cysteine.

Numerous studies show that resistant human cancer cell lines contain high glutathione levels in vitro and that oxidant-induced toxicity can be enhanced by buthionine sulfoximine (BSO) that causes glutathione depletion by inhibiting its synthesis. The role of glutathione in oxidant and drug resistance has not been previously investigated in mesothelioma.

Enzymes in the glutathione redox cycle: Glutathione peroxidase (GPx) and glutathione reductase (GR)

GPx is one of the major enzyme families in removing hydrogen peroxide generated by, e.g., superoxide dismutases. It catalyzes the reaction where GSH is oxidized into GSSG and H2O2 converted into water and oxygen. Four distinct selenoproteins are included in the family of glutathione peroxidases, the classical form being the cytosolic GPx, which is also found in the mitochondria and extracellular matrix.

The other three are the gastrointestinal form of GPx, a non-selenium dependent GPx and phospholipid hydroperoxide GPx. The cytosolic GPx, a tetrameric selenoprotein, has a molecular weight of 85 000. The gene is in chromosome 3. Recently a polymorphism was found that associated with lung cancer.

In normal physiological conditions with low or moderate production of H2O2, GPx has been considered a more important scavenger than catalase, because its Michaelis-Menten constant (Km value) for H2O2 is lower than that of catalase. Selenium is needed in the synthesis of GPx and at least the extracellular GPx is induced by hyperoxia and oxidants.

GPx is ubiquitously expressed in erythrocytes, kidney, and liver. The expression of GPx in malignant tumors is somewhat variable. GPx activity has been suggested to be elevated in adenocarcinoma of the lung. Whereas it was decreased in other lung cancer subtype biopsies when assessed by immunohistochemistry.

Elevated GPx activity has been linked with chemoresistance of anticancer drugs, such as adriamycin, that kills cells by releasing free radicals. Glutathione reductase (GR) converts GSSG back to GSH at the expense of NADPH forming a redox cycle. Two isoenzymes of GR, one cytosolic and one mitochondrial, are encoded by a single gene located in chromosome 8.

It has been postulated that the glutathione conjugates formed in xenobiotic detoxification can inhibit GR thereby accumulating GSSG altering the redox capacity of the cell. The expression of GR in human lung and tumors is unclear.

Enzymes in glutathione biosynthesis

-glutamylcysteine synthetase (GCS) is the rate-limiting enzyme in GSH biosynthesis. The synthesis requires another ATP-dependent enzyme, glutathione synthase, and the amino acids glutamic acid, cysteine and glycine. In general, the activity of GCS defines the rate of glutathione synthesis and GCS is feedback-inhibited by the product, GSH.

Cysteine is the rate-limiting substrate. Levels of GSH and cysteine are the two factors that regulate the synthesis of glutathione under physiological conditions. The importance of glutathione synthesis was proven in a recent the study which showed that homozygous knockout mouse lacking the GCS heavy subunit gene dies before birth.

GCS is a cytosolic heterodimer consisting of a heavy subunit (GCSh, MW~ 73 000) and a light subunit GCSl, MW ~30 000. GCSh gene is in chromosome 6 (6p12) and GCSl gene in chromosome 1 (1p21) and two mRNA transcripts are consistently seen for both subunits. GCSh is the catalytically active subunit; it also binds the feedback inhibitor GSH.

It has been suggested that GCSh alone comprises about half of the enzyme activity when compared with the holoenzyme. Some studies, however, have concluded that it has no catalytic activity without the light subunit. GCSl serves as an important regulatory role and reduces the inhibitory effect of GSH.

It has been suggested that during GSH depletion, in oxidizing conditions, the enzyme undergoes conformational changes between subunits that allow an increase in the enzyme activity. In normal physiological conditions when abundant amounts of GSH are present, both subunits are needed for the enzyme activity. GCS is induced by several agents, including oxidants e.g. H2O2 and menadione, cytokines e.g. TNF, heavy metals e.g. cadmium and iron, and some chemotherapeutic agents e.g. cisplatin.

At transcriptional level, GCS subunits are regulated by several regulatory signals, including ARE, TRE, AP1 and NF-B. GCS activity is also regulated at the post-transcriptional and translational level, and phosphorylation/dephosphorylation may control its activity. Possible inhibitors include glucocorticoids, insulin, prostaglandin E, and TGF-.

Exposure to sublethal doses of oxidants may initiate an adaptive antioxidant response, where the intracellular GSH is first depleted leading to oxidant stress and consequent GCS upregulation. The role of -glutamyl transpeptidase in regulating GCS activity indirectly by cleaving extracellular GSH has also been suggested. The expression of GCS mRNA varies between different tissues.

In healthy human lungs, GCS mRNA has been detected from bronchial epithelial cells. There are no previous studies on the expression or distribution of GCS in malignant tumors. It has been suggested that chromosome 1 (loss of 1p21-22) is often deleted in malignant mesothelioma.

This would predispose an individual to the development of the tumor. Elevated levels of GCS have been detected in many drug-resistant malignant cell lines. Chemoresistance may be associated with the accumulation of GSH, which functions as an antioxidant but is also used in detoxification reactions.

Glutathione has also been shown to inhibit apoptosis by changing the redox state of the cell. Apoptosis resistance, in turn, has been considered important in the drug resistance of malignant cells. Glutathione synthase (GS) is a cytosolic homodimer that catalyzes the reaction of L- -glutamyl-Lcysteine and glycine that forms GSH.

GS is composed of two apparently identical subunits (each MV~52 000) and the gene is in chromosome 20. Two forms of glutathione synthetase deficiency have been described. One form is mild, causing hemolytic anemia, but the other more severe form causes 5-oxo-prolinuria with secondary neurological involvement. The regulation of GS is poorly known.

In glutathione biosynthesis, the availability of cysteine is crucial. Cysteine is transported into the cell by a sodium-dependent A system and cystine, an oxidized form of cysteine, by an inducible transporter Xc-. Cystine is then reduced to cysteine that can be used in GSH biosynthesis. The transport of cystine is induced by oxidants, such as hyperoxia and H2O2, contributing to increased GSH levels during oxidative stress.

There are no studies on the expression of GS or cysteine transporters in malignant tumors. glutamyl transpeptidase (GT) acts as a salvage enzyme in GSH synthesis. The molecular weight is 50 kD for the heavy and 25 kD for the light subunit (Arai et al., 1995). The gene is in chromosome 22. GT is located on the plasmamembrane, where it cleaves the -glutamyl bond in extracellular -glutamyl cysteinyl-glycine.

The amino acids are returned into the cell and reused for GSH synthesis. GT is induced by menadione and t-butyl hydroquinone, suggesting its role in protecting cells during oxidative stress. In addition to other luminal surfaces of the body, lung epithelium contains high levels of this enzyme.

 There is one study showing that mesothelioma biopsies are negative for this enzyme when assessed by immunohistochemistry. In the same study, strong immunoreactivity is detected from renal cell carcinoma, adenocarcinoma of the prostate and papillary carcinoma of the thyroid.

Glutathione S-transferases (GSTs)

GSTs are a superfamily of detoxifying enzymes that have broad substrate specificitie. Five families of cytosolic GSTs have been identified in humans, of which four have been thoroughly characterized: Alpha, Mu, Pi, and Theta. The genes for GST-class are all located in chromosome 1, whereas GST-the gene is in chromosome 11. A polymorphism of GSTM1 (-class) resulting in dysfunction of the enzyme has proven to be a risk factor for malignant diseases, including mesothelioma. 

The GSTs conjugate GSH with compounds containing an electrophilic center and thereby provide critical protection against xenobiotics and products of oxidative stress. Since the GSH-conjugate is transported out of the cell, intracellular GSH is consumed irreversibly in the conjugation and thus maintenance of intracellular GSH levels is essential for the optimal function of GSTs. 

Many GST enzymes possess GPx activity as well. Many of the substrates of GSTs also induce the expression of the GST genes, suggesting an adaptive response to chemical stress. Carcinogens and alkylating agents may induce GST-. 

The GST-ð family is the predominant GST in human solid tumors and has even been used as a marker in lung, colon, bladder and other human cancers (Zhang et al., 1998). GST activity is often associated with anticancer drug resistance, as the drugs are converted to a less toxic form by the conjugation. Based on one study 77% of mesothelioma cell lines expressed GST in immunohistochemistry.

Catalase

Catalase (CAT) is a tetrameric hemoprotein that catalyses the reaction of decomposition of H2O2 into water and oxygen. It has a molecular weight of 240 000. It is mainly localized in the peroxisomes (Davies et al., 1979) but is also found in the cytoplasm and mitochondria in minor amounts. The gene is localized in chromosome 11. Patients suffering from acatalasemia have a mutation of the CAT gene but are clinically healthy.

Catalase has a higher Km than GPx, which suggests a major role for CAT at higher levels of H2O2 but a minor role at physiological levels of H2O2. Catalase is not abundantly present in the mitochondria, where the physiological oxidative stress is at its highest. It has been shown to be induced by high oxygen tension in alveolar epithelial cells. 

In other studies, however, no induction could be detected in lung epithelial cells after oxidant or cytokine exposures. There are no systematic studies on catalase in malignant tumors. Some studies have suggested variable catalase expression in lung, breast and colon cancers. One recent study showed that catalase is highly expressed in mesothelioma. No major role has been suggested to catalase in drug resistance.

Other proteins with antioxidant capacity

Glutaredoxin and peroxiredoxins are cysteine-containing H2O2-scavenging proteins, that have been recently described, but no investigations of these proteins have been conducted in human lung tumors. Thioredoxin is composed of two closely related cysteinecontaining proteins, thioredoxin (TRX) and thioredoxin reductase (TRXR).

This group of proteins enhances cell proliferation and increases resistance to apoptosis in several in vitro and in vivoexperimental models. There are two recent studies showing overexpression of TRX and TRXR in lung tumors and mesothelioma (Kahlos et al., 2001a; Soini et al., 2001a). However, the expression of these proteins did not correlate with survival in either tumor. Heme oxygenase (heat shock protein 32) has also been shown to have antioxidative properties.