The opening chapter of this book asked what is the origin of genetic diseases that reduce fertility and so are not maintained by children inheriting from parental sufferers, but which nevertheless do not die out and sometimes increase in prevalence. We described these as one-generation genetic diseases. These diseases include Down's syndrome, a substantial part of all diabetes, epilepsy, fragile X syndrome and many kinds of mental and physical handicap. The risk of these diseases increases with parental mutation rates.

Most research on mutation is financed by cancer research funds but amongst the studies published are many describing mutation in germ cells and discussing environmental mutagens. Study has expanded to include antimutagens, which are substances that destroy mutagens or inhibit their effects. Diet may be more or less mutagenic, or, in the words of a study from Ames (1983): "Diet is a source of mutagens and antimutagens". Where are mutagens to be found and can they be avoided? Where are antimutagens to be found and are they a defence? A report from the International Commission says (ICPEMC, 1986):

"In view of the widespread occurrence of mutagenic compounds in food and beverages as well as in other parts of the environment, effective elimination of exposure to many genotoxic chemicals is not feasible."

The report explains that it is not referring only to mutagenic chemicals in the environment of our industrial society such as lead or various constituents of smoke but also to the naturally occurring mutagens including oxygen radicals produced in food by oxidation for example of some fatty acids. In the context of reproduction a diet that is too mutagenic is a bad diet. The possibility of defence against the mutagens in the environment by antimutagens in food introduces a new scale of good and bad. The search for antimutagens is worldwide and expanding (Knudsen, 1986). The International Commission commented:

"One group of likely candidates for safe use in mutation and cancer prevention are natural constituents in our diet. These may at present actually determine the level of protection against genotoxic compounds. This applies to vitamins A, C and E and also to selenium in areas of low selenium intake."

The intake of foods and drinks may be changed to increase the antimutagenicity of a daily diet. This is relevant to choice of diet during the days and weeks before and around conception when the risk is high of new mutations, which are almost invariably harmful. It was noted in Chapter Four that a deficiency of protein and of particular vitamins and minerals could increase mutation rate. The role of some nutrients like vitamins A, C and E and of minerals like selenium in making some mutagens less potent or harmless may be of consequence even when diets cannot be described as deficient. Mutagens and antimutagens can modulate mutagenicity whether or not a diet is adequate by conventional criteria. Apart from some vitamins and minerals the antimutagens chemically identified so far include many less familiar plant constituents notably phenolic compounds, including some tannins and flavonoids, ellagic acid and gallic acid, sulfhydryl compounds including glutathione, carotenes, some terpenes, indoles and cyanates (JCPEMC, 1986).

The present chapter discusses mutagens and antimutagens in diet within the digestive tract. Only the part of the diet absorbed makes a contribution to mutation rate and this contribution is very variable depending upon the contents of the digestive tract. Vitamins A, E, betacarotene and vitamin C which survive digestion and are absorbed continue their antioxidant role within the body. Among the important antimutagens within the body are the heme containing proteins which include the mutagen-metabolizing enzymes of the liver. These antimutagens contain iron which is best provided by heme in the diet. The antimutagenicity of heme is illustrated in Figure 6.1. Among the defences of every body cell against mutagens is the active enzyme glutathione peroxidase, which requires the essential nutrient selenium. A shortage of selenium can cause a raised mutation rate (Hayatsu et at., 1988; ICPEMC, 1986). These endogenous antimutagens are synthesized within the body and their synthesis requires an efficient supply of all the essential nutrients needed for protein synthesis including efficient intracellular energy metabolism. There are, however, many antimutagens in food that are not classified as essential nutrients.

THE ANTIMUTAGENICITY OF FRESH VEGETABLES AND FRUIT

Most of the known antimutagens are naturally occurring constituents of plants including familiar vegetables and fruits. Kada and his colleagues of the Department of Induced Mutation of the Japanese Institute of Genetics have made systematic searches for such constituents by measuring the inhibiting effect of plant juices on the mutagenic action in bacteria of the pyrolysis products of amino acids produced by, for example, cooking meat at excessively high temperatures (Kada et al., 1978; Kada, 1982). The pyrolysis of protein produces a range of mutagens of great potency, many of which have now been chemically identified. Among the antimutagenic plant juices first identified were those of cabbage, ginger, radish and turnip. The results for cabbage juice are summarized in Table 6.1. The mutagen used in this study, Try-P, was produced by the pyrolysis of the amino acid tryptophan. It is seen in Table 6.1 that the antimutagens in cabbage were destroyed by boiling for 30 minutes, while the mutagen was not destroyed. The antimutagens in cabbage were not identified and were not in the cabbage fibre but in the juice and were not precipitated by a centrifuge. Many of the antimutagens in other vegetables are also destroyed by boiling.

TABLE 6.1

THE ANTIMUTAGENICITY OF CABBAGE JUICE AND ITS DESTRUCTION BY BOWING; AMES TEST USING TRY-P AS MUTAGEN

treatment	mutations per plate
no cabbage juice, no mutagen	44
cabbage juice, but no mutagen	41
mutagen and raw cabbage juice	51
mutagen and boiled cabbage juice	280
mutagen only	295

Source: Kada et al., 1978; Ames et al., 1975

Fifty-nine different vegetables and fruits were examined by Morita et al. (1978), also from the Japanese Institute of Genetics, and they found that all except 2 were in some degree antimutagenic, again using mutagens produced by the heating of amino acids. Thirty-six of these vegetables and fruits are listed in Table 6.2 in order of their antimutagenicity against a single mutagen, again Try-P.

TABLE 6.2

THE COMPARATIVE ANTIMUTAGENICITY OF SOME FRESH FOODS; PERCENTAGE OF MUTAGEN INACTIVATED

burdock	95		turnip leaf	55
mint leaf	93		taro	51
broccoli	91		garlic	48
green pepper	91		banana	47
apple	87		pears	46
shallot	87		rape blossom	42
pineapple	87		rape blossom	41
ginger	85		Chinese cabbage	35
cabbage	83		Welsh onion	34
vaubergine	81		lettuce	29
beefsteak plant	78		pumpkin	29
parsley	78		celery	27
cauliflower	76		yarn	23
grapes	74		turnip root	15
sweet potato	65		green asparagus	10
radish	59		red cabbage	8
chicory	58		cucumber	5
oranges	55		field pea	1

Source: Morita et al., 1978.

Burdock (arctium lappa) at the top of the list is cultivated as a vegetable in Japan and young leaves, stems and roots are eaten. Taro (Cobcasia esculenta) is a root vegetable of Asian origin with many varieties widely used in tropical countries. The full list in Morita92s paper included other Japanese vegetables not sold by the British greengrocer, but none of great antimutagenic merit although some Japanese mushrooms are of interest. The ranking of fresh foods, as illustrated in Table 6.2, is different for other mutagens and each antimutagen has its own spectrum. Some antimutagens have a wide spectrum and some a narrow. For example tested against the mutagenic pyrolysis products of 4 other amino acids burdock, broccoli and aubergine were found to be effective antimutagens for all 4, while apples were effective against 3 and cabbage against only one. The limited spectrum of most if not all antimutagens is a great complication and is characteristic not only of food extracts like apple juice but of chemical constituents like vitamin C.

Most of the new antimutagenic compounds currently being discovered are plant constituents, but many of the most effective are heat-labile and difficult to isolate and have been shown in particular cases to be specific proteins and enzymes. More than a 100 specific chemicals have been identified as antimutagens. The difficulty of deciding on any application of the research on antimutagens may be illustrated from the research on tea which is produced from the leaves of came/ha sinensis. Over 70 per cent of the soluble matter in tea consists of tannins which are derivatives of the compound catechin. One particular catechin (epigallocatechin gallate) making up to 5 per cent of green tea powder has been found to be a very potent antimutagen (Kada et al. 1985). It is a long way from such a discovery to establishing the range of mutagens and biological environments in which green tea extracts are effective. However such a discovery is to be taken seriously because tea is used by around one half the world92s population and catechins are reasonably stable compounds.

How far are the antimutagens in foods a defence against mutagens found in the environment? Nitroso compounds are an important class of mutagens that occur naturally, but have been increased in the human environment by pollution of water with nitrates from agricultural fertilizer and by some methods of food preservation used in the manufacture of ham and bacon, some other preserved meats, some kinds of cheese, smoked products and some cosmetics. Smoke including vehicle exhaust gases contains oxides of nitrogen that can be precursors of these mutagens. Two precursors of nitroso compounds were used as mutagens in experiments by Barale et al. (1983) of the University of Pisa. The antimutagenicity of carrots, cauliflower, lettuce, spinach and strawberries is illustrated in Figures 6.2 and 6.3 based on this research.

Male mice were used in the experiments and yeast was used for measuring mutagenic activity. These experiments illustrate the antimutagenicity of some fresh foods for a particular class of mutagen. Figure 6.2 is based on measurements of mutagenicity of the contents of the digestive tract and shows the antimutagenicity of 5 juices. Many antimutagens act in this way, preventing the absorption of mutagens and also preventing mutation in the lining of the digestive tract. Figure 6.3 based on intrahepatic measurements shows that the 5 juices continued to be effective against nitroso compounds after absorption, but with a different order of antimutagenic potency. In both experiments juices were used and not whole vegetables so that the antimutagens must have been in the juices and not in the fibre.

Many mutagens cause mutations by reacting chemically with DNA for example by alkylation. Yano (1979) of Saitama Medical School, investigated the effect of the juice of 7 vegetables and milk on the mutagenicity of alkylating mutagens. The 7 vegetable juices decomposed these mutagens and made them non-mutagenic in different degrees as illustrated in Table 6.3. The potency of milk in inhibiting alkylation was greater than that of the vegetable juices on a volume basis. Milk has however a higher solid content than the vegetable juices, so Yano recalculated potency using an arbitrary scale based on freeze-dried material and it was then comparable with the fresh vegetable juices.

A growing list of desirable, but not essential, antimutagenic nutrients is being added to the lists of essential nutrients accepted as necessary for survival. Chlorophyll, the green colouring constituent of plants essential for photosynthesis, is an example of a desirable nutrient. It was reported in 1979 from the University of Texas that chlorophyll appeared to be the most active antimutagenic substance in wheat sprouts (Lai et al, 1980). It has been shown in many subsequent studies that chlorophyll, or its salt chlorophyllin, is an antimutagen with an exceptionally broad spectrum. Ong et al. (1986, 1989) of the US National Institute of Occupational Safety and Health compared the potency of chlorophyllin, retinol, beta-carotene, vitamin C and vitamin E as anti-mutagens. Figure 6.4 illustrates the antimutagenic effect of chlorophyllin on fried minced pork.

Ong prepared other mixtures of mutagens from substances to which it was thought that many people are often exposed, including eoal dust, diesel engine emission particles, airbome dust particles, tobacco snuff and beef over-cooked at 230^oC. Chiorophyllin was 69 per cent effective against the tobacco snuff and 90 per cent against the 4 other complex mixtures. Retinol was 29-48 per cent effective against all 5 mixtures, beta-carotene somewhat less so and vitamins C and F almost ineffective. Beta-carotene, although a precursor of retinol, is not itself an essential nutrient, but is an antimutagen and desirable nutrient that is entering large scale production by the drug industry in USA. The Ames test used by Ong can do no more than identify possibly useful substances and the paper concluded:

"It seems that chiorophyllin is potentially useful for the prevention of health hazards that may be caused by genotoxic agents."

The antimutagencity of chlorophyll may be another reason why it is wise to green vegetables, but assessment of the practical value of this knowledge remains to be done together with similar information about other antimutagens.

The B vitamins are important antimutagens. Benzpyrene is a mutagenic constituent of smoke and it is seen in Figure 6.5 based on Dutch research that riboflavin is an antimutagen for benzpyrene and also for cigarette smoke condensate (Terwel & van der Hoeven, 1985). B vitamins are essential for DNA synthesis and repair, and it has been shown, for example, that niacin reduces oxygen radical induced dam

THE ANTIMUTAGENICITY OF SOME FATS AND OILS

The overall mutagenic status of a diet depends on the potency and concentration of mutagens and antimutagens it contains. Unsaturated fatty acids provide an important part of the antimutagenicity of most diets. The most ubiquitous fatty acid with the highest concentration in most foods is oleic, which is mono-unsaturated. The antimutagenicity of oleic and other fatty acids has been studied by Hayatsu and colleagues of the Faculty of Pharmaceutical Sciences, Okayama University (Hayatsu et al., 1981a, 1981b. 1988). The antimutagenicity of oleic acid is illustrated for one mutagen in Figure 6.6, but oleic acid is effective in the inhibition of a broad spectrum of mutagens including not only protein pyrolysis compounds, but nitroso compounds and polycyclic hydrocarbons.

Olive oil contains from 65 to 85 per cent of oleic acid and an average of 11 per cent of linoleic acid, the most ubiquitous polyunsaturated fatty acid which is an even more potent antimutagen than oleic. The potency of different vegetable oils as antimutagens varies widely, the most potent being safflower seed, sunflower seed, soyabean oil and corn oil in that order. Coconut or palm oil contain substantial percentages of saturated fatty acids which have no effect on mutagenic status. Of the grains oats has the highest percentage of unsaturated fatty acids, 16 per cent of its energy value depending on these unsaturated fats compared with less than 5 per cent for wheat. The antimutagenicity of some dozen unsaturated fatty acids has been studied and they have all been found to be antimutagenic in different degress. However Hayatsu (1981a, 198 ib) reported that the ether extract of normal human faeces was antimutagenic and that oleic and linoleic acids were the effective inhibitors. The full significance of this finding is still not clear, but it seems that one role of these unsaturated fatty acids is the maintenance of the antimutagenicity of the whole contents of the digestive tract. New mutagens are generated during digestion so consumption of antimutagens is desirable apart from the need to neutralize mutagens in the diet. Meals based too heavily on some carbohydrates may contain too little unsaturated fat, although some whole grains notably oats contain adequate fat of excellent quality.

Any commendation of unsaturated fats for their antimutagenicity applies only to fats that have not been heated in the course of cooking or food processing above the temperature of boiling water. If unsaturated fats are overheated by themselves they produce a series of mutagenic products. However when fat is overheated in cooking it is usually associated with meat or fish or eggs or other food rich in protein. The fat is either an inherent part of the food or is added as in frying.

MUTAGENS PRODUCED BY COOKING AT HIGH TEMPERATURES

Highly mutagenic substances are produced by heating protein and these mutagens appear to be responsible for most of the mutagenicity of the average diet. A study of the Dutch diet, supported by both Dutch and American government agencies found that much of the mutagenicity of the average Dutch diet was caused by cooking (Alink et al., 1988). Food was prepared with a composition matching average Dutch food consumption. A first set of pellets was made from raw components before cooking and had only a low mutagenicity, indeed had no detectable mutagenicity in the first test. The same raw food "after processing under usual household conditions (at approximately 150^oC)" was clearly mutagenic in the Ames test used in this study. Addition of fruit and vegetables reduced but did not eliminate the mutagenicity of the cooked food pellets. Three of the mutagens responsible for the mutagenicity were identified and were found to be the same as previously identified in fried beef by other research workers. This Dutch study had many obvious limitations. Many antimutagens apparently act by energizing digestive enzymes and a mixture of foods in laboratory tests would not for this reason alone be expected to show the same mutagenicity as in the digestive tract. The choice of 1 50^oC as an average cooking temperature in Dutch kitchens may be correct but mutagen production increases rapidly above this temperature which is often exceeded both in the home and in commercial cooking.

As part of a study at the University of California seven "fast food" restaurants offering grilled hamburgers were visited and it was reported (Bjeldanes et al., 1982):

"Of the seven restaurants we sampled two provided hamburgers with consistently high mutagen content, three with consistently low mutagen content, and two showed considerable variation between sampling times. Precise data on the cooking times and temperatures of the hamburgers are not available. However, discussion with each vendor indicated a qualitative correlation of higher mutagen content with more severe cooking conditions, including the use of increased temperature associated with the sporadic high values occurring when the cook was rushed."

The hamburgers in this study varied in mutagenicity by a factor of over 50 in the Ames test. The mutagenicity of beef, pork or eggs increases as the cooking temperature increases from 150^oC to 3000 C and they become charred. Mutagenicity increases with cooking time as well as temperature and may vary by a factor of 100 or more. The Dutch experiments excluded mutagens produced at these higher temperatures. It has been shown that mutagens are produced in beef and beef extract by boiling at 1000 C (Commoner, 1978). However the mutagens at 100^oC are negligible compared with those produced at the higher temperatures. Boiling or braising protein around 100^oC does not make a serious contribution to the mutagenicity of any diet.

The digestive tract provides a first line of defence against the ingress of mutagens. If the contents of the human digestive tract were always antimut agenic doubts about the desirability of overheating protein might be unjustified. Many mutagens are wholly or partly destroyed during digestion. Small samples of people from Canada, Japan, South Africa, USA and elsewhere show that some people have antimutagenic faeces and that for them any absorption of mutagens from the digestive tract is presumably very small. However there are people who have mutagenic, even highly mutagenic, faeces, and mutagens may then be absorbed. The first line of defence against mutagens can be breached. Baker et al. (1982) showed that about one-third of the potent mutagenic activity in fried bacon and pork reappeared in the urine of human subjects very quickly after they were eaten. The increase in the mutagenicity of the urine by a factor of 10 or more persisted for about 12 hours and then declined to normal in another 12 hours. The meat was not charred but fried in its own fat at temperatures of 150-190^oC. Sjodin and Jagerstad (1984) reported that fried meat mutagens fed to rats are partly absorbed and that the urine then becomes mutagenic. Hayatsu et al. (1985) showed that mutagens from fried meat eaten by humans made their urine mutagenic. Hayatsu commented:

"Our results on the effect of beef ingestion are consistent with the pork and bacon results in terms of the acute nature of the response. The present study has demonstrated that this phenomenon can take place under normal, everyday eating conditions".

Accordingly Hayatsu examined the mutagenicity of urine of volunteers after their everyday meals. Figure 6.7 shows for one man the mutagenicity of urine lingering after a fried beef lunch and 6.8 for the same man after a fried beef dinner. Lindeskog et al. (1988) of the Karolinska Institute, Stockholm, has shown experimentally in rats that if fried meat is consumed every day the mutagenicity of faeces particularly, but also of urine, is cumulative and increases for at least a week with a diurnal cycle. Mutagens from fried meat are carried by the blood to all body systems and are possible mutagenic agents for the germ line. One of the beef protein pyrolysis products has been shown to be a potent mutagen in hamster ovary cells (Thompson et al., 1987). Sugimura and Nagao of the National Cancer Center, Toyko, as long ago as 1982 said that it was not yet possible to decide on the comparative importance of different mutagens but nevertheless:

"Based on recent information it seems wise to reduce exposure to these mutagens as much as possible. This would probably be possible without disturbing the quality of life. For instance, it would be simple to avoid charring food."

Three years later Sugimura said there was "no doubt about the necessity" of reducing the formation and consumption of mutagens in cooked food (Sugimura, 1985).

REDUCING THE MUTAGENICITY WITHIN THE DIGESTIVE TRACT

Dion et al. (1982) of the Ludwig Institute of Cancer Research, Toronto, has shown the effect on faecal mutagenicity of a dietary supplement of 400mg of both vitamin C and vitamin E daily. Vitamin C and E are both antioxidants. These vitamins may not be such effective antimutagens as chlorophyll or have such a wide spectrum but they have both been shown to inhibit the formation of the mutagenic nitroso compounds or to destroy them (Newmark & Mergens, 1981; Bruce et al., 1979; Newberne & Suphakam, 1983). The variation from day-to-day in the faecal mutagenicity of a single donor on a controlled diet or on a controlled diet with a supplement of vitamins C and E is shown in Figure 6.9.

It is seen that the faecal mutagenicity declined steadily from the first of the 15 days when the vitamins were taken and the decline was particularly rapid during the first 3 days. There was a 3 week interval between the taking of the controlled diet without the two vitamin supplements and the 15 days that included supplements. Examination of faeces does, of course, show only the end result of the conflict between mutagens and antimutagens higher up the digestive tract. Figure 6.9 suggests that vitamins C and E have the capacity to reduce digestive tract mutagenicity in some cases. The effect of vitamins C and E on the digestive tract mutagenicity of 19 donors is compared in Figure 6.10.