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Should the concept of the recommended dietary allowance be altered to incorporate interactive effects of ubiquitous pollutants?
Medical
Hypotheses
5:
1273-1285,
1979
SHOULD THE CONCEPT OF THE RECOMXENDED DIETARY ALLOWANCE BE ALTERED TO INCO~O~TE INTE~CTIVE EFFECTS OF UBIQUITOUS POLLUT~TS?
Edward J. Calabrese, Division of Public Health, University of Massachusetts, U.S.A. Amherst, Massachusetts, 01003 ABSTRACT Using ascorbic acid as a model, this paper proposes that the concept of the RDA should be broadened to take into account the effects of ubiquitous pollutants on human health, a factor presently not incorporated in RDA derivations. It is now widely accepted that ascorbic acid nutritional status markedly affects the toxicity and/or carcinogenicity of greater than 50 pollutants, many of which are ubiquitous in the air, water, and food environments. At the present time, the data do not warrant changing the ascorbic acid RDA in light of the knowledge of pollutant interactions. Key Words:
ascorbic acid, RDA, smoking, nitrosamine, carcinogen, environment. INTRODUCTION
Recommended Dietary Allowances (RDAs) are levels of intake of essential nutrients considered, in the judgement of the Food and Nutrition Board on the basis of available scientific knowledge, "to be adequate to meet the known nutritional needs of practically all healthy persons" (1). The data upon which the RDA is based include how much of the nutrient is needed to satisfy growth, maintain body weight, prevent loss of nutrients from the body, sustain specific physiological functions and prevent specific signs of a deficiency. The RDA is designed to estimate the level of substance that will satisfy the vitamin or mineral requirements in the vast majority of the population, that is 97.5 per cent of the population or the mean plus two standard deviations. The RDA does not include the potential increased needs which may be required for those persons undergoing severe stress or disease above the typical minor infections and stresses which a "normal" existence experiences. Thus, the RDA is not designed as a standard for "high risk
1273
groups" who have unusally high nutrient needs because of either environmental or genetic reasons (1). Furthermore, the RDA is only concerned with nutritional effects as opposed to pharmacologic functions. This is an important distinction since they may occur at vastly different levels (i.e. orders of magnitude different). Harper (2) states that a nutrient function may be characterized by the amount of nutrient required to prevent the appearance of signs and symptoms caused by a lack of the nutrients or some metabolic reflection of this need, and not to use alleviation of some condition (e.g. the serum-cholesterol lowering effect of niacin) that does not result from a lack of nutrient as the criterion. It is the intention of this paper to evaluate the hypothesis that the concept of RDA should be modified to incorporate a specific consideration for the enhanced stress of certain ubiquitous environmental contaminants in humans. This proposal will be supported by demonstrating that a) nutritional status markedly affects pollutant toxicity/carcinogenicity as well as pollutant exposure affecting nutritional status, and b) the RDAs have not specifically addressed the issue of exposure to ubiquitous pollutants. Finally, as a model for this investigation, the RDA of ascorbic acid will be evaluated in light of pollutant-vitamin interactions. Interaction of Ascorbic Acid and Toxic Substances Numerous researchers have evaluated the influence of ascorbic acid on pollutant toxicity in a variety of animal models including humans. The extent of such nutrient-pollutant interactions are enormous with well over 50 such interactions having been reported. Table 1 provides a partial list of pollutants whose toxicity is affected by ascorbic acid treatment or whose administration affects ascorbic acid nutritional status (3). Of these interactions of ascorbic acid with environmental pollutants, approximately a dozen have sufficient evidence to cause concern for the public health and/or industrial health. Several of the most important associations with ascorbic acid include those with cadmium, chromium (hexavalent), nitrites, nitrosames, and smoking. Other important although less documented associations with ascorbic acid include chlorinated hydrocarbon insecticides, fluoride, lead and mercury, PCBs, vanadium, and endogenous aromatic amines (3). Sxamples of two ascorbic acid-pollutant associations may serve to illustrate the significance of such interactions. Smoking and Ascorbic Acid One of the most realistic and quantifiable threats of environmental factors affecting nutritional status is that of smoking. Large scale studies have demonstrated that persons who smoke equal to or greater than 20 cigarettes per day exhibit a 40 per cent reduction in median serum vitamin C levels as compared to non-smokers (4). When these smokers and non-smokers were matched for comparable vitamin C intake similar findings were obtained. Median serum
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TABLE I Partial List of Toxic Substances Whose Toxicity and/or Carcinogenicity is Modified by Ascorbic Acid
A.
Pesticides 1. Several Chlorinated Hydrocarbons Insecticides 2. Several Organophosphate Insecticides
B.
Heavy 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
C.
Selected Hydrocarbons Used In Industry 1. Benzanthrone 2. Benzene 3. Chloroform 4. Glycerol 5. Hydrazine 6. Polychlorinated Biphenyls 7. TNT 8. Vinyl Chloride
D.
Gaseous Pollutants 1. Carbon Monoxide 2. Ozone
Source:
Metals Arsenic Cadmium Chromium Cobalt Copper Cyanide Fluoride Lead Mercury Selenium Silica Tellurium
E.J. Calabrese (1979). Nutrition and Environmental Health: The Influence of Nutritional Status on Pollutant Toxicity andCarcinogenicity. Volume 1. The Vitamins. John Wiley and Sons, Inc. Publishers. New York, NY.
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vitamin C levels of both male and female smokers in Canada who consumed between 0 to 30 mg of ascorbic acid per day were quite low, especially the values for males from Quebec which were only 0.22 mg per cent, a value suggestive of subclinical scurvy. Since this value represents a median, it follows that a considerable number of smokers in this vitamin C consumption group have values even lower than 0.22 mg per cent (4). The importance of these findings lie in several domains: (a) millions of people (i.e. smokers) are affected; (b) p ersons who consume inadequate or even marginally adequate diets may be actually getting much less vitamin C than recommended and (c) that cigarette smoke contains appreciable levels of cadmium. It would seem that smokers, therefore, may find it necessary to supplement their typical diets with ascorbic acid to ensure that recommended levels are achieved :4). Nitrosamine Formation One may occasionally hear the comment made that if one consumes food treated with sodium nitrite be sure to drink some orange juice so that the accompanying ascorbic acid may prevent the formation of the animal carcinogen, nitrosamine. An excellent attempt to quantify human exposure within the gastrointestinal tract from nitrosamines has been presented by Mirvish (5). Based on the rate constant of dimethylnitrosamine (DMN) formation at the pH of 3.4, he calculated the quantity of this carcinogen which theoretically should be formed in the gastric fluids following consumption of foods with realistic levels of nitrite and dimethylamine. Thus, if a person ingested a 300 g meal which contained 12 mg of dimethylamine hydrochloride and 60 mg of sodium nitrite (i.e. a relatively realistic situation),* no greater than 3 ug of DMN would be predicted to form. Since this is the ideal stochiometric outcome one would expect considerably less than the 3 ug formation. However, the evidence is quite clear and seemingly irrefutable that ascorbic acid prevents the nitrosation of secondary amines so that nitrosamine formation is reduced. When the ascorbic acid is present in a 2:l molar ratio to nitrite, Since the it is capable of preventing the formation of nitrosamines. agerage American is thought to ingest 1.5 to 4.1 mg of nitrite per day (6), it would appear that about 3.0 to 8.2 mg of ascorbic acid on the average would be needed to prevent the formation of the nitrosamines. However, average values are somewhat misleading and may not be of value in assessing specific situations. For example, as was previously noted, a typical 300 g meat or fish meal with 200 ug sodium nitrate/g of food will provide approximately 60 mg of nitrite, thereby suggesting that upwards of 120 mg of ascorbic acid would be needed to prevent any formation of the nitrosamines. *Mirvish (5) noted that a fish protein concentrate was reported to have 152 mg/kg of mixed amine hydrochlorides of which l/4 was the In addition, sodium nitrite has been used dimethylamine hydrochloride. as a fish and meat preservative at levels up to 200 ug/g of food.
1276
Since the formation of nitrosamines occurs prior to absorption, it is apparent that if one obtained all his ascorbic acid at breakfast but consumed nitrite plus secondary amines at later meals, the ascorbic acid which was consumed at breakfast would have long since left the stomach. Thus, no protection would be available from ascorbic acid to prevent the nitrosation of the secondary amines. It would seem logical that if one is consuming food with nitrite that a good policy would include the concomitant consumption of extra amounts of ascorbic acid as previously estimated. Since the ascorbic acid molecule outcompetes the amine for the nitrosating agent, it should be assumed that each nitrosated ascorbic acid molecule although preventing the formation of the nitrosamine is also prevented from functioning as a normal ascorbic acid molecule and may not even be absorbed. Consequently, the presence of nitrite should also increase the individual's requirement of ascorbic acid. Recent data of Librojo and Hatchcock (7) support this interpretation. It should be emphasized that other dietary substances can also prevent the formation of nitrosamines including tannic acid (from drinking tea) (8), vitamin E (9), and other substances (10). These results are based on either animal studies or -in vitro simulations of the human gastric fluid and not on direct human experimentation. However, the evidence is so consistent and striking that it appears legitimate to assume that humans may act similarly to the animal models at least until future studies discredit the presently accepted models. The RDA For Ascorbic Acid - Its Rationale In light of the above suggestion to ingest ascorbic acid during meals that contain nitrites as well as all the known and proposed associations of ascorbic acid with environmental/occupational pollutants, the question naturally arises as to whether the RDA should be modified to include a consideration of the role of ascorbic acid in reducing the toxicity and carcinogenicity of many substances. The studies from which the RDA for ascorbic acid was derived generally concerned themselves with (a) ensuring the prevention of scurvy, (b) providing an adequate safety factor for a highly heterogeneous population and (c) calculating the amount of ascorbic acid needed to ensure body pool repletion and/or tissue saturation. These studies were not specifically designed to consider the influence of pollutant exposureonascorbic acid nutritional status or how ascorbic acid may have affected pollutant toxicity. It may be argued by some that exposure to a wide variety of substances was implicitly considered in every study since all diets must have had definite pollutant levels. Since toxicity from these pollutants was never reported it seemed logical that the levels of ascorbic acid were sufficient to prevent adverse effects. Consequently, extra supplementation of ascorbic acid is not necessary. This perspective is strongly supported by the fact that the studies of Barker, et al, (11) and Hodges, et al, (12) upon which the recent recommended RDA of the NAS (45 mg) is based actually utilized all smokers. Since 30 mg per day was able to maintain a full body pool in these persons,
1277
one could assume that even less ascorbic acid would be needed to ensure a full body pool for non-smokers [see Harper, (2)]. This reasoning may be challenged in part on several points. These researchers did not specifically evaluate the influence of pollutants on the health of people with variable levels of ascorbic acid in their diets. Not only weren't pollutant levels monitored, neither were the health indicators of such exposures. Thus, to claim that the individuals who were studied appeared not to be adversely affected by any pollutants is not a logical conclusion. Should the RDA Provide Protection Against Environmental Toxicants? It has been reasoned that if the body pool of ascorbic acid is replete as is known to occur at an approximate daily intake of 45 mg (Hodges, et al, 12,13), then there is no need or value in consuming anymore. In fact, excess consumption would only be rapidly excreted and, therefore, wasteful. Thus, it is assumed that ascorbic acid performs its functions after its gastrointestinal absorption. Amongst the previously mentioned protective actions which ascorbic acid displays as part of what appears to be normal functions, there are several important detoxication type activities which appear to be so called non-vitamin functions. These may include the inhibition of the nitrosation of secondary amines, the reduction of hexavalent chromium to the trivalent form, and the proposed prevention of the formation of oxidized metabolities of endogenous aromatic amines within the bladder. In fact, Schlegal (14) states that he recommended that his patients who are at increased risk to developing bladder cancer should consume upwards of 1.5 g of ascorbic acid daily in order to maintain a relatively high concentration of ascorbic acid within the urine. Yet, the action of ascrobic acid in the gastrointestinal tract and in the urine would not normally be considered as vitamin functions and thus not included in the derivation of the RDA. Whether the presence of elevated levels of ascorbic acid in the urine of so-called non-susceptibles would also offer protection from aromatic amine induced bladder cancer was not addressed. It may be argued that the RDA is designed to ensure protection for the normal person and not specifically the person at increased risk to developing bladder cancer or the industrial worker exposed to elevated levels of particular pollutants. Yet, it must not be overlooked that many of the pollutants that may have their toxicity reduced by ascorbic acid are ubiquitous. For instance, the average American is thought to ingest approximately 70 ug of cadmium per day (15), 0.9 mg arsenic per day (16,17), 600 to 900 p Ci Ra226 per year (18), 1.5 to 4.1 mg NO3 per day (6), 35 mg DDT per year (19,20) as well as ambient air exposures to ozone, CO, and cigarette smoke. Thus, the typical American is constantly exposed to a wide diversity of toxic substances each day of their life. Of course, each person's exposure will vary according to geographical location, as well as cultural and dietary habits. Yet, exposure to multiple pollutants is inevitable.
1278
These exposures may, of course, be superimposed on (a) whether one's occupation provides additional exposure to toxic substances, (b) whether oral contraceptives are used (c) whether medication such as aspirin and tetracycline are taken and (d) whether one smokes tobacco. The Case for Supplementation When discussing the validity of the RDA for ascorbic acid, the issue of excess supplementation must also be addressed. It is generally accepted that once tissues become saturated with vitamin C the excess gets excreted. Thus, of what value in fighting pollutant effects would supplements be if they were in excess of what is needed to saturate tissues? Of significance was the report by Wagstaff and Street (21) which indicated that levels of vitamin C in the diet (25 ppm) of guinea pigs which could prevent scurvy were not capable of inducing microsomal enzyme induction following dietary exposure to dieldrin. Even an ascorbic acid level of 50 ppm (2 mg/day) did not appreciably increase enzyme induction. However, at 200 ppm (7-8 mg/day) or 8 times greater than the amount which prevented scurvy (i.e., 25 ppm) occurred a 50 percent increase in enzyme induction while at 2000 ppm (70 mg/day) ascorbic acid in the diet, the capacity for dieldrin to induce enzyme synthesis increased to 80 percent above the 50 ppm level.* In light of the fact that humans ingest food with appreciable residues of chlorinated hydrocarbons such as DDT (35 mg/year), these findings may have profound implications. At the least, they suggest a possible inadequacy of the present RDA since it appeared that a beneficial function occurred at daily exposures greater than necessary to saturate tissues. Gobovich and Maistruk (23) also pointed out that 150 mg/day enhance< the excretion of excess flouride in industrial workers as compared to those given 50 and 100 mg/day. In addition, Varghese, et al., (24) have shown that supplementation of 4 grams of ascorbic acid each day can reduce by 70 percent the nitroso compounds normally found in feces. Similar decreases in the number of bacterial revertants were also found, thereby confirming the protective effects of vitamin C with respect to the presumed formation of N-nitroso rompounds. Conclusions At the present time the knowledge of how ascorbic acid influences pollutant toxicity and/or carcinogenicity is extremely interesting and promising but generally too incomplete upon which to base specific policy directives. For nearly every pollutant/ascorbic acid interaction, either definitive studies supporting such relationships remain to be conducted or * Reid (22) reported that a daily intake of 50 mg or more of ascorbic acid was needed for tissue saturation in the guinea pig.
1279
the dose-response protective relationships are insufficiently quantified. For instance, although the data on the influence of smoking on ascorbic acid status is very convincing, it still may be legitimately criticized because no sufficiently detailed methodology (including food preparation techniques, analysis of food samples, etc.) quantifying ascorbic acid intake has been encorporated into any study. This criticism is even more severe with respect to the proposed oral contraceptive effects on ascorbic acid. The Soviet studies on noise, flax dust, and fluoride lack a comprehensive handling of almost all the confounding variables which may affect ascorbic acid nutritional status (3). Furthermore, studies with animals which synthesize their own ascorbic acid (i.e. rats, mice) provide only suggestive evidence as to how the human would respond. For example, data indicating that ascorbic acid supplemention in rats significantly reduces PCB toxicity when the dietary ratio of ascorbic acid to PCB was 2O:l (25) do not permit one to simplistically calculate that since an average American may consume 10 ug PCB/day (26) that 200 ug of ascorbic acid in the diet should offset the toxic effects from PCBs. In addition to assessing the most precisely defined relationships, it is important not to overlook areas of research which are potentially very exciting but yet lack sufficient data. For example, early studies have suggested that ascorbic acid may reduce benzene toxicity. Unfortunately, comprehensive studies to assess this important relationship have not been attempted. This is extremely relevant today especially in light of the suggestion that benzene is a carcinogen. Other areas of research should be directed toward the potential effects of ascorbic acid on the development of silicosis, noise related adverse health effects, industrial fluoride toxicity, lead intoxication, and the adverse effects of vanadium exposure. These are areas which, for the most part (except for lead), have just been briefly considered (3). As this discussion on vitamin C concludes the following points should be recognized: 1.
Ascorbic acid nutritional status markedly affects the toxicity of numerous environmental stressors including both chemical and physical agents. In the vast majority of cases the toxicity is reduced.
2.
Even though ascorbic acid may be capable of facilitating the detoxification of a wide variety of toxicants and carcinogens, it may also enhance the toxicity of several compounds. a.
Recent studies have indicated that ascorbic acid may facilitate the absorption of inorganic and organic mercury and lead from the gastrointestinal tract in guinea pigs Such findings suggest that and rats, respectively. elevated dietary levels of ascorbic acid may enhance the toxicity of mercurial and lead compounds (27,28).
1280
b.
Megadoses of ascorbic acid (3 g/day) are known to markedly reduce body stores of cystpine and may enhance toxicity of cyanide and other compounds requiring the conjugating properties of this sulphur containing amino acid (31). These studies strongly imply that caution should be be employed before advocating the consumption of large supplementary doses of ascorbic acid.
3.
A variety of environmental stressor agents (e.g. smoking) can significantly affect the ascorbic acid nutritional status.
4.
The incidence of gastric cancer is inversely associated with the consumption of diets high in ascorbic acid; at least one animal carcinogen (i.e. nitrosamines) may be prevented from being formed in the gastrointestinal tract in the presence of ascorbic acid.
5.
Many of the pollutants which have their toxicity and/or carcinogenicity reduced by ascorbic acid are ubiquitous in our environment including air, water, and food.
6.
The present RDA of 60 mg has not taken into account evidence that ascorbic acid dietary status can markedly reduce the toxicity of many substances.* Although it is assumed that once body stores are replete with ascorbic acid (presumably from the ingestion of 60 mg/day) that all requirements for ascorbic acid should be satisfied, evidence has been presented from Soviet researchers that consumption of 150 mg/day can enhance excess flouride excretion. Whether supplementation in excess of what is needed to saturate tissues will further enhance the detoxification of pollutants is an important issue which needs to be addressed more fully.
*As indicated earlier, the present RDA was based on research with smokers. Consequently, the present RDA does specifically address and make provision for the apparent fact that smoking reduces the absorption of ascorbic acid into the blood stream.
1281
7.
Many actions of ascorbic acid occur "outside" the body, that is, in the gastrointestinal tract and possibly in the urinary bladder. However, these functions are usually considered non-vitamin roles of ascorbic acid.
8.
The concept of RDA should be broadened to include the protective interaction of ascorbic acid with ubiquitous pollutants. At the present time, the data do not warrant changing the RDA in light of knowledge of pollutant interactions.** However, future studies concerning pollutant interaction could, in fact, cause an increase in the RDA.
9.
The role of nutritional toxicity should be more fully implemented in occupational health policies and practices. Industries should educate workers on the role of good nutrition in preventing pollutant induced occupational disease. It is further suggested that OSHA establish pilot programs designed to provide nutritional monitoring of workers in industries where the adverse effects of pollutants may be modified by nutritional status. This is an area totally ignored by OSHA in its attempt to protect the worker. OSHA has focused only on the elimination of the stressor agents and, unfortunately, has ignored enhancing the adaptive capacity of the worker. Neither goal is mutually exclusive of the other, although it is true that certain industries may use the concept of proper nutrition to perpetuate a dirty workplace in order to save money (and not health:).
10.
Research needs in environmental/occupational health should include a strong orientation toward nutrient-pollutant interactions. Of all the areas of life that government can affect this is one which is much more readily accessable to human control and, therefore, potential improvement.
**That the present RDA may be inadequate was suggested by C.G. King (see Harper, (2), discussion) who stated that "there is evidence that the level now recommended may not be enough to give maximum protection against stresses, such as infection or exposure -to toxic materials."
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REFERENCES 8th edit. National Academy of SC iences.
1.
Recommended Dietary Allowances. Washington, D.C. 1974.
2.
Harper AE. The recommended dietary allowances for ascorbic acid. Ann. N.Y. Acad. Sci. 258:491-497.
3.
Calabrese EJ. !$utrition and Environmental Health: Influence of Nutritional Volume 1. The Vitamins. Status on Pollutant Toxicity and Carcinogenicity. .John Wiley and Sons, Inc. Publishers. New York, NY.
4.
Pelletier 0. Vitamin C and cigarette smokers. 258~156-167, 1975.
5.
Mirvish SS. Kinetics of dimethylamine nitrosation in relation to J. Nat. Cancer Inst. 44:633, 1970. nitrosamine carcinogenesis.
6.
White JW. Relative significance of dietary sources of nitrate and nitrite. J. Agri. Food Chem. 23:886, 1975.
7.
Librojo NT, Hatchcock JN. Nitrite effects on ascorbic acid metabolism in guinea pigs and rats. Presented at the 12th Annual Conference on Trace Substances in Environmental Health. June 5-6, 1978, University of Missouri, Columbia, 1978.
8.
Mirvish SS. Blocking the formation of n-nitroso compounds with ascrobic acid -in vitro and -in vivo. Ann. N.Y. Acad. Sci. 258:175-180, 1975.
9.
Kamm JJ, Dashman T, Newmark H, Mergens WJ. Inhibition of amine-nitrite hepatotoxicity by a-tocopherol. Toxicol. Appl Pharmacol. 41:575-583, 1977.
Ann. N.Y. Acad. Sci. -
10.
Klubes P, Cerna I, Rabinowitz AD, et al. Factors affecting DMN form intestinal bacteria. Food Cosmet. Toxicol. 10~757-767, 1972.
11.
Baker EM, Hodges RE, Hood J, Sauberlich, HE, March SC, Canham JE. Metabolism of C- and H- labelled L-ascorbic acid in human scurvy. Amer. J. Clin. Nutr. 24:444-454, 1971.
12.
Hodges RE, Hood J, Canham JE, Sauberlich HE, Baker EM. Clinical manifestations of ascorbic acid deficiency in man. Amer. J. Clin. _ Nutrit. 24~432-443, 1971.
13.
Hodges PE, Baker EM, Hood J, Sauberlich HE, March SC. Experimental scurvy in man. Amer. J. Clin. Nutrit. 22:535-548, 1969.
14.
Schlegel JU. Proposed uses of ascorbic acid in prevention of bladder carcinoma. Ann. N.Y. Acad. Sci. 258~432-437, 1975.
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15.
Fox MR. Nutritional influences on cadmium toxicity. Perspect. (In press), 1979.
16.
Spencer H, Lewin I, Fowler .I, Samachson J. calcium absorption and balances in man. 22(4):381-390, 1969.
17.
Calabrese EJ. Methodological Approaches to Deriving Environmental and Occupational Health Standards. Wiley-Interscience, N.Y. pp 162-165, 1978.
18.
Comar CL, Rust JH. Natural radioactivity in the biosphere and foodstuffs. In: Toxicants Occurring Naturally in Foods. 2nd Edition. National Academy of Sciences. Washington, D,C, pp 95-97, 1973.
19.
Kraybill HF. Significance of pesticide residues in food in relation to total environmental stress. Can. Med. Assoc. Jour. 100:204-215, 1969.
20.
Kraybill HF. Conceptual approaches to the assessment of non-occupational environmental cancer. In: Advances in Modern Toxicology. Vol. 3, Environmental Cancer. Kraybill, HF, Mehlman MA. John Wiley and Sons, N.Y. p. 32, 1977.
21.
Wagstaff DJ, Street JC. Ascorbic acid deficiency and induction of hepatic microsomal hydroxylative enzymes by organochlorine pesticides. Toxicol. Appl. Pharmacol. 19:10-19, 1971.
22.
Reid ME. Nutrient requirements of the guinea pigs. Nutrient requirements of laboratory animals number X. A report of the committee on Animal Nutrition. National Academy of Sciences. National Research Council. pp 11-23, 1962.
23.
Gabovich RD. Maistruk PN. On the therapeutic and prophylactic diet in the fluroine manufacturing industry. Vopr@sy Pitaniia 22:32-38, 1963.
24.
Varghese AJ, Land PC, Furrer R, Bruce WR. Non-volatile n-nitroso compounds in human feces. In: Environmental aspects of n-nitroso compounds. International Agency for Research on Cancer. IARC Scientific Publications. No. 19, 257-264, 1978.
25.
Chakraborty D, et al. Biochemical studies on polychlorinated biphenyl toxicity in rats: manipulation by vitamin C. Internat. J. Vit. Nutr. Res. 8(1):22-31, 1978.
26.
Calabrese EJ, Sorensen AJ. The health effects of PCBs with particular emphasis on human high risk groups. Rev. on Environ. Health. 2(4): 285-304, 1977.
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Environ. Health
Effect of sodium flouride on Amer. J. Clin. Nutr.
27.
Blackstone S, Hurley RI, Hughes RE. Some interrelationship between Food vitamin C (L-ascorbic acid) and mercury in the guinea pig. -and Cosmetic Toxicology. 12:511-516, 1974.
28.
Conrad ME, Barton JC. Factors affecting the absorption and excretion of lead in the rat. Gasteroenterology 74~731-740, 1978.
29.
Basu TK. acid.
Possible toxicological aspects of megadoses of ascorbic 16:247-250, 1977. Chem-Biol. Interactions.
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Hypotheses
5:
1273-1285,
1979
SHOULD THE CONCEPT OF THE RECOMXENDED DIETARY ALLOWANCE BE ALTERED TO INCO~O~TE INTE~CTIVE EFFECTS OF UBIQUITOUS POLLUT~TS?
Edward J. Calabrese, Division of Public Health, University of Massachusetts, U.S.A. Amherst, Massachusetts, 01003 ABSTRACT Using ascorbic acid as a model, this paper proposes that the concept of the RDA should be broadened to take into account the effects of ubiquitous pollutants on human health, a factor presently not incorporated in RDA derivations. It is now widely accepted that ascorbic acid nutritional status markedly affects the toxicity and/or carcinogenicity of greater than 50 pollutants, many of which are ubiquitous in the air, water, and food environments. At the present time, the data do not warrant changing the ascorbic acid RDA in light of the knowledge of pollutant interactions. Key Words:
ascorbic acid, RDA, smoking, nitrosamine, carcinogen, environment. INTRODUCTION
Recommended Dietary Allowances (RDAs) are levels of intake of essential nutrients considered, in the judgement of the Food and Nutrition Board on the basis of available scientific knowledge, "to be adequate to meet the known nutritional needs of practically all healthy persons" (1). The data upon which the RDA is based include how much of the nutrient is needed to satisfy growth, maintain body weight, prevent loss of nutrients from the body, sustain specific physiological functions and prevent specific signs of a deficiency. The RDA is designed to estimate the level of substance that will satisfy the vitamin or mineral requirements in the vast majority of the population, that is 97.5 per cent of the population or the mean plus two standard deviations. The RDA does not include the potential increased needs which may be required for those persons undergoing severe stress or disease above the typical minor infections and stresses which a "normal" existence experiences. Thus, the RDA is not designed as a standard for "high risk
1273
groups" who have unusally high nutrient needs because of either environmental or genetic reasons (1). Furthermore, the RDA is only concerned with nutritional effects as opposed to pharmacologic functions. This is an important distinction since they may occur at vastly different levels (i.e. orders of magnitude different). Harper (2) states that a nutrient function may be characterized by the amount of nutrient required to prevent the appearance of signs and symptoms caused by a lack of the nutrients or some metabolic reflection of this need, and not to use alleviation of some condition (e.g. the serum-cholesterol lowering effect of niacin) that does not result from a lack of nutrient as the criterion. It is the intention of this paper to evaluate the hypothesis that the concept of RDA should be modified to incorporate a specific consideration for the enhanced stress of certain ubiquitous environmental contaminants in humans. This proposal will be supported by demonstrating that a) nutritional status markedly affects pollutant toxicity/carcinogenicity as well as pollutant exposure affecting nutritional status, and b) the RDAs have not specifically addressed the issue of exposure to ubiquitous pollutants. Finally, as a model for this investigation, the RDA of ascorbic acid will be evaluated in light of pollutant-vitamin interactions. Interaction of Ascorbic Acid and Toxic Substances Numerous researchers have evaluated the influence of ascorbic acid on pollutant toxicity in a variety of animal models including humans. The extent of such nutrient-pollutant interactions are enormous with well over 50 such interactions having been reported. Table 1 provides a partial list of pollutants whose toxicity is affected by ascorbic acid treatment or whose administration affects ascorbic acid nutritional status (3). Of these interactions of ascorbic acid with environmental pollutants, approximately a dozen have sufficient evidence to cause concern for the public health and/or industrial health. Several of the most important associations with ascorbic acid include those with cadmium, chromium (hexavalent), nitrites, nitrosames, and smoking. Other important although less documented associations with ascorbic acid include chlorinated hydrocarbon insecticides, fluoride, lead and mercury, PCBs, vanadium, and endogenous aromatic amines (3). Sxamples of two ascorbic acid-pollutant associations may serve to illustrate the significance of such interactions. Smoking and Ascorbic Acid One of the most realistic and quantifiable threats of environmental factors affecting nutritional status is that of smoking. Large scale studies have demonstrated that persons who smoke equal to or greater than 20 cigarettes per day exhibit a 40 per cent reduction in median serum vitamin C levels as compared to non-smokers (4). When these smokers and non-smokers were matched for comparable vitamin C intake similar findings were obtained. Median serum
1274
TABLE I Partial List of Toxic Substances Whose Toxicity and/or Carcinogenicity is Modified by Ascorbic Acid
A.
Pesticides 1. Several Chlorinated Hydrocarbons Insecticides 2. Several Organophosphate Insecticides
B.
Heavy 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
C.
Selected Hydrocarbons Used In Industry 1. Benzanthrone 2. Benzene 3. Chloroform 4. Glycerol 5. Hydrazine 6. Polychlorinated Biphenyls 7. TNT 8. Vinyl Chloride
D.
Gaseous Pollutants 1. Carbon Monoxide 2. Ozone
Source:
Metals Arsenic Cadmium Chromium Cobalt Copper Cyanide Fluoride Lead Mercury Selenium Silica Tellurium
E.J. Calabrese (1979). Nutrition and Environmental Health: The Influence of Nutritional Status on Pollutant Toxicity andCarcinogenicity. Volume 1. The Vitamins. John Wiley and Sons, Inc. Publishers. New York, NY.
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vitamin C levels of both male and female smokers in Canada who consumed between 0 to 30 mg of ascorbic acid per day were quite low, especially the values for males from Quebec which were only 0.22 mg per cent, a value suggestive of subclinical scurvy. Since this value represents a median, it follows that a considerable number of smokers in this vitamin C consumption group have values even lower than 0.22 mg per cent (4). The importance of these findings lie in several domains: (a) millions of people (i.e. smokers) are affected; (b) p ersons who consume inadequate or even marginally adequate diets may be actually getting much less vitamin C than recommended and (c) that cigarette smoke contains appreciable levels of cadmium. It would seem that smokers, therefore, may find it necessary to supplement their typical diets with ascorbic acid to ensure that recommended levels are achieved :4). Nitrosamine Formation One may occasionally hear the comment made that if one consumes food treated with sodium nitrite be sure to drink some orange juice so that the accompanying ascorbic acid may prevent the formation of the animal carcinogen, nitrosamine. An excellent attempt to quantify human exposure within the gastrointestinal tract from nitrosamines has been presented by Mirvish (5). Based on the rate constant of dimethylnitrosamine (DMN) formation at the pH of 3.4, he calculated the quantity of this carcinogen which theoretically should be formed in the gastric fluids following consumption of foods with realistic levels of nitrite and dimethylamine. Thus, if a person ingested a 300 g meal which contained 12 mg of dimethylamine hydrochloride and 60 mg of sodium nitrite (i.e. a relatively realistic situation),* no greater than 3 ug of DMN would be predicted to form. Since this is the ideal stochiometric outcome one would expect considerably less than the 3 ug formation. However, the evidence is quite clear and seemingly irrefutable that ascorbic acid prevents the nitrosation of secondary amines so that nitrosamine formation is reduced. When the ascorbic acid is present in a 2:l molar ratio to nitrite, Since the it is capable of preventing the formation of nitrosamines. agerage American is thought to ingest 1.5 to 4.1 mg of nitrite per day (6), it would appear that about 3.0 to 8.2 mg of ascorbic acid on the average would be needed to prevent the formation of the nitrosamines. However, average values are somewhat misleading and may not be of value in assessing specific situations. For example, as was previously noted, a typical 300 g meat or fish meal with 200 ug sodium nitrate/g of food will provide approximately 60 mg of nitrite, thereby suggesting that upwards of 120 mg of ascorbic acid would be needed to prevent any formation of the nitrosamines. *Mirvish (5) noted that a fish protein concentrate was reported to have 152 mg/kg of mixed amine hydrochlorides of which l/4 was the In addition, sodium nitrite has been used dimethylamine hydrochloride. as a fish and meat preservative at levels up to 200 ug/g of food.
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Since the formation of nitrosamines occurs prior to absorption, it is apparent that if one obtained all his ascorbic acid at breakfast but consumed nitrite plus secondary amines at later meals, the ascorbic acid which was consumed at breakfast would have long since left the stomach. Thus, no protection would be available from ascorbic acid to prevent the nitrosation of the secondary amines. It would seem logical that if one is consuming food with nitrite that a good policy would include the concomitant consumption of extra amounts of ascorbic acid as previously estimated. Since the ascorbic acid molecule outcompetes the amine for the nitrosating agent, it should be assumed that each nitrosated ascorbic acid molecule although preventing the formation of the nitrosamine is also prevented from functioning as a normal ascorbic acid molecule and may not even be absorbed. Consequently, the presence of nitrite should also increase the individual's requirement of ascorbic acid. Recent data of Librojo and Hatchcock (7) support this interpretation. It should be emphasized that other dietary substances can also prevent the formation of nitrosamines including tannic acid (from drinking tea) (8), vitamin E (9), and other substances (10). These results are based on either animal studies or -in vitro simulations of the human gastric fluid and not on direct human experimentation. However, the evidence is so consistent and striking that it appears legitimate to assume that humans may act similarly to the animal models at least until future studies discredit the presently accepted models. The RDA For Ascorbic Acid - Its Rationale In light of the above suggestion to ingest ascorbic acid during meals that contain nitrites as well as all the known and proposed associations of ascorbic acid with environmental/occupational pollutants, the question naturally arises as to whether the RDA should be modified to include a consideration of the role of ascorbic acid in reducing the toxicity and carcinogenicity of many substances. The studies from which the RDA for ascorbic acid was derived generally concerned themselves with (a) ensuring the prevention of scurvy, (b) providing an adequate safety factor for a highly heterogeneous population and (c) calculating the amount of ascorbic acid needed to ensure body pool repletion and/or tissue saturation. These studies were not specifically designed to consider the influence of pollutant exposureonascorbic acid nutritional status or how ascorbic acid may have affected pollutant toxicity. It may be argued by some that exposure to a wide variety of substances was implicitly considered in every study since all diets must have had definite pollutant levels. Since toxicity from these pollutants was never reported it seemed logical that the levels of ascorbic acid were sufficient to prevent adverse effects. Consequently, extra supplementation of ascorbic acid is not necessary. This perspective is strongly supported by the fact that the studies of Barker, et al, (11) and Hodges, et al, (12) upon which the recent recommended RDA of the NAS (45 mg) is based actually utilized all smokers. Since 30 mg per day was able to maintain a full body pool in these persons,
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one could assume that even less ascorbic acid would be needed to ensure a full body pool for non-smokers [see Harper, (2)]. This reasoning may be challenged in part on several points. These researchers did not specifically evaluate the influence of pollutants on the health of people with variable levels of ascorbic acid in their diets. Not only weren't pollutant levels monitored, neither were the health indicators of such exposures. Thus, to claim that the individuals who were studied appeared not to be adversely affected by any pollutants is not a logical conclusion. Should the RDA Provide Protection Against Environmental Toxicants? It has been reasoned that if the body pool of ascorbic acid is replete as is known to occur at an approximate daily intake of 45 mg (Hodges, et al, 12,13), then there is no need or value in consuming anymore. In fact, excess consumption would only be rapidly excreted and, therefore, wasteful. Thus, it is assumed that ascorbic acid performs its functions after its gastrointestinal absorption. Amongst the previously mentioned protective actions which ascorbic acid displays as part of what appears to be normal functions, there are several important detoxication type activities which appear to be so called non-vitamin functions. These may include the inhibition of the nitrosation of secondary amines, the reduction of hexavalent chromium to the trivalent form, and the proposed prevention of the formation of oxidized metabolities of endogenous aromatic amines within the bladder. In fact, Schlegal (14) states that he recommended that his patients who are at increased risk to developing bladder cancer should consume upwards of 1.5 g of ascorbic acid daily in order to maintain a relatively high concentration of ascorbic acid within the urine. Yet, the action of ascrobic acid in the gastrointestinal tract and in the urine would not normally be considered as vitamin functions and thus not included in the derivation of the RDA. Whether the presence of elevated levels of ascorbic acid in the urine of so-called non-susceptibles would also offer protection from aromatic amine induced bladder cancer was not addressed. It may be argued that the RDA is designed to ensure protection for the normal person and not specifically the person at increased risk to developing bladder cancer or the industrial worker exposed to elevated levels of particular pollutants. Yet, it must not be overlooked that many of the pollutants that may have their toxicity reduced by ascorbic acid are ubiquitous. For instance, the average American is thought to ingest approximately 70 ug of cadmium per day (15), 0.9 mg arsenic per day (16,17), 600 to 900 p Ci Ra226 per year (18), 1.5 to 4.1 mg NO3 per day (6), 35 mg DDT per year (19,20) as well as ambient air exposures to ozone, CO, and cigarette smoke. Thus, the typical American is constantly exposed to a wide diversity of toxic substances each day of their life. Of course, each person's exposure will vary according to geographical location, as well as cultural and dietary habits. Yet, exposure to multiple pollutants is inevitable.
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These exposures may, of course, be superimposed on (a) whether one's occupation provides additional exposure to toxic substances, (b) whether oral contraceptives are used (c) whether medication such as aspirin and tetracycline are taken and (d) whether one smokes tobacco. The Case for Supplementation When discussing the validity of the RDA for ascorbic acid, the issue of excess supplementation must also be addressed. It is generally accepted that once tissues become saturated with vitamin C the excess gets excreted. Thus, of what value in fighting pollutant effects would supplements be if they were in excess of what is needed to saturate tissues? Of significance was the report by Wagstaff and Street (21) which indicated that levels of vitamin C in the diet (25 ppm) of guinea pigs which could prevent scurvy were not capable of inducing microsomal enzyme induction following dietary exposure to dieldrin. Even an ascorbic acid level of 50 ppm (2 mg/day) did not appreciably increase enzyme induction. However, at 200 ppm (7-8 mg/day) or 8 times greater than the amount which prevented scurvy (i.e., 25 ppm) occurred a 50 percent increase in enzyme induction while at 2000 ppm (70 mg/day) ascorbic acid in the diet, the capacity for dieldrin to induce enzyme synthesis increased to 80 percent above the 50 ppm level.* In light of the fact that humans ingest food with appreciable residues of chlorinated hydrocarbons such as DDT (35 mg/year), these findings may have profound implications. At the least, they suggest a possible inadequacy of the present RDA since it appeared that a beneficial function occurred at daily exposures greater than necessary to saturate tissues. Gobovich and Maistruk (23) also pointed out that 150 mg/day enhance< the excretion of excess flouride in industrial workers as compared to those given 50 and 100 mg/day. In addition, Varghese, et al., (24) have shown that supplementation of 4 grams of ascorbic acid each day can reduce by 70 percent the nitroso compounds normally found in feces. Similar decreases in the number of bacterial revertants were also found, thereby confirming the protective effects of vitamin C with respect to the presumed formation of N-nitroso rompounds. Conclusions At the present time the knowledge of how ascorbic acid influences pollutant toxicity and/or carcinogenicity is extremely interesting and promising but generally too incomplete upon which to base specific policy directives. For nearly every pollutant/ascorbic acid interaction, either definitive studies supporting such relationships remain to be conducted or * Reid (22) reported that a daily intake of 50 mg or more of ascorbic acid was needed for tissue saturation in the guinea pig.
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the dose-response protective relationships are insufficiently quantified. For instance, although the data on the influence of smoking on ascorbic acid status is very convincing, it still may be legitimately criticized because no sufficiently detailed methodology (including food preparation techniques, analysis of food samples, etc.) quantifying ascorbic acid intake has been encorporated into any study. This criticism is even more severe with respect to the proposed oral contraceptive effects on ascorbic acid. The Soviet studies on noise, flax dust, and fluoride lack a comprehensive handling of almost all the confounding variables which may affect ascorbic acid nutritional status (3). Furthermore, studies with animals which synthesize their own ascorbic acid (i.e. rats, mice) provide only suggestive evidence as to how the human would respond. For example, data indicating that ascorbic acid supplemention in rats significantly reduces PCB toxicity when the dietary ratio of ascorbic acid to PCB was 2O:l (25) do not permit one to simplistically calculate that since an average American may consume 10 ug PCB/day (26) that 200 ug of ascorbic acid in the diet should offset the toxic effects from PCBs. In addition to assessing the most precisely defined relationships, it is important not to overlook areas of research which are potentially very exciting but yet lack sufficient data. For example, early studies have suggested that ascorbic acid may reduce benzene toxicity. Unfortunately, comprehensive studies to assess this important relationship have not been attempted. This is extremely relevant today especially in light of the suggestion that benzene is a carcinogen. Other areas of research should be directed toward the potential effects of ascorbic acid on the development of silicosis, noise related adverse health effects, industrial fluoride toxicity, lead intoxication, and the adverse effects of vanadium exposure. These are areas which, for the most part (except for lead), have just been briefly considered (3). As this discussion on vitamin C concludes the following points should be recognized: 1.
Ascorbic acid nutritional status markedly affects the toxicity of numerous environmental stressors including both chemical and physical agents. In the vast majority of cases the toxicity is reduced.
2.
Even though ascorbic acid may be capable of facilitating the detoxification of a wide variety of toxicants and carcinogens, it may also enhance the toxicity of several compounds. a.
Recent studies have indicated that ascorbic acid may facilitate the absorption of inorganic and organic mercury and lead from the gastrointestinal tract in guinea pigs Such findings suggest that and rats, respectively. elevated dietary levels of ascorbic acid may enhance the toxicity of mercurial and lead compounds (27,28).
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b.
Megadoses of ascorbic acid (3 g/day) are known to markedly reduce body stores of cystpine and may enhance toxicity of cyanide and other compounds requiring the conjugating properties of this sulphur containing amino acid (31). These studies strongly imply that caution should be be employed before advocating the consumption of large supplementary doses of ascorbic acid.
3.
A variety of environmental stressor agents (e.g. smoking) can significantly affect the ascorbic acid nutritional status.
4.
The incidence of gastric cancer is inversely associated with the consumption of diets high in ascorbic acid; at least one animal carcinogen (i.e. nitrosamines) may be prevented from being formed in the gastrointestinal tract in the presence of ascorbic acid.
5.
Many of the pollutants which have their toxicity and/or carcinogenicity reduced by ascorbic acid are ubiquitous in our environment including air, water, and food.
6.
The present RDA of 60 mg has not taken into account evidence that ascorbic acid dietary status can markedly reduce the toxicity of many substances.* Although it is assumed that once body stores are replete with ascorbic acid (presumably from the ingestion of 60 mg/day) that all requirements for ascorbic acid should be satisfied, evidence has been presented from Soviet researchers that consumption of 150 mg/day can enhance excess flouride excretion. Whether supplementation in excess of what is needed to saturate tissues will further enhance the detoxification of pollutants is an important issue which needs to be addressed more fully.
*As indicated earlier, the present RDA was based on research with smokers. Consequently, the present RDA does specifically address and make provision for the apparent fact that smoking reduces the absorption of ascorbic acid into the blood stream.
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7.
Many actions of ascorbic acid occur "outside" the body, that is, in the gastrointestinal tract and possibly in the urinary bladder. However, these functions are usually considered non-vitamin roles of ascorbic acid.
8.
The concept of RDA should be broadened to include the protective interaction of ascorbic acid with ubiquitous pollutants. At the present time, the data do not warrant changing the RDA in light of knowledge of pollutant interactions.** However, future studies concerning pollutant interaction could, in fact, cause an increase in the RDA.
9.
The role of nutritional toxicity should be more fully implemented in occupational health policies and practices. Industries should educate workers on the role of good nutrition in preventing pollutant induced occupational disease. It is further suggested that OSHA establish pilot programs designed to provide nutritional monitoring of workers in industries where the adverse effects of pollutants may be modified by nutritional status. This is an area totally ignored by OSHA in its attempt to protect the worker. OSHA has focused only on the elimination of the stressor agents and, unfortunately, has ignored enhancing the adaptive capacity of the worker. Neither goal is mutually exclusive of the other, although it is true that certain industries may use the concept of proper nutrition to perpetuate a dirty workplace in order to save money (and not health:).
10.
Research needs in environmental/occupational health should include a strong orientation toward nutrient-pollutant interactions. Of all the areas of life that government can affect this is one which is much more readily accessable to human control and, therefore, potential improvement.
**That the present RDA may be inadequate was suggested by C.G. King (see Harper, (2), discussion) who stated that "there is evidence that the level now recommended may not be enough to give maximum protection against stresses, such as infection or exposure -to toxic materials."
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2.
Harper AE. The recommended dietary allowances for ascorbic acid. Ann. N.Y. Acad. Sci. 258:491-497.
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Calabrese EJ. !$utrition and Environmental Health: Influence of Nutritional Volume 1. The Vitamins. Status on Pollutant Toxicity and Carcinogenicity. .John Wiley and Sons, Inc. Publishers. New York, NY.
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Pelletier 0. Vitamin C and cigarette smokers. 258~156-167, 1975.
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Mirvish SS. Kinetics of dimethylamine nitrosation in relation to J. Nat. Cancer Inst. 44:633, 1970. nitrosamine carcinogenesis.
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White JW. Relative significance of dietary sources of nitrate and nitrite. J. Agri. Food Chem. 23:886, 1975.
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Librojo NT, Hatchcock JN. Nitrite effects on ascorbic acid metabolism in guinea pigs and rats. Presented at the 12th Annual Conference on Trace Substances in Environmental Health. June 5-6, 1978, University of Missouri, Columbia, 1978.
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Mirvish SS. Blocking the formation of n-nitroso compounds with ascrobic acid -in vitro and -in vivo. Ann. N.Y. Acad. Sci. 258:175-180, 1975.
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Kamm JJ, Dashman T, Newmark H, Mergens WJ. Inhibition of amine-nitrite hepatotoxicity by a-tocopherol. Toxicol. Appl Pharmacol. 41:575-583, 1977.
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Klubes P, Cerna I, Rabinowitz AD, et al. Factors affecting DMN form intestinal bacteria. Food Cosmet. Toxicol. 10~757-767, 1972.
11.
Baker EM, Hodges RE, Hood J, Sauberlich, HE, March SC, Canham JE. Metabolism of C- and H- labelled L-ascorbic acid in human scurvy. Amer. J. Clin. Nutr. 24:444-454, 1971.
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Hodges RE, Hood J, Canham JE, Sauberlich HE, Baker EM. Clinical manifestations of ascorbic acid deficiency in man. Amer. J. Clin. _ Nutrit. 24~432-443, 1971.
13.
Hodges PE, Baker EM, Hood J, Sauberlich HE, March SC. Experimental scurvy in man. Amer. J. Clin. Nutrit. 22:535-548, 1969.
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Schlegel JU. Proposed uses of ascorbic acid in prevention of bladder carcinoma. Ann. N.Y. Acad. Sci. 258~432-437, 1975.
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Fox MR. Nutritional influences on cadmium toxicity. Perspect. (In press), 1979.
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Spencer H, Lewin I, Fowler .I, Samachson J. calcium absorption and balances in man. 22(4):381-390, 1969.
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Calabrese EJ. Methodological Approaches to Deriving Environmental and Occupational Health Standards. Wiley-Interscience, N.Y. pp 162-165, 1978.
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Comar CL, Rust JH. Natural radioactivity in the biosphere and foodstuffs. In: Toxicants Occurring Naturally in Foods. 2nd Edition. National Academy of Sciences. Washington, D,C, pp 95-97, 1973.
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Kraybill HF. Significance of pesticide residues in food in relation to total environmental stress. Can. Med. Assoc. Jour. 100:204-215, 1969.
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Kraybill HF. Conceptual approaches to the assessment of non-occupational environmental cancer. In: Advances in Modern Toxicology. Vol. 3, Environmental Cancer. Kraybill, HF, Mehlman MA. John Wiley and Sons, N.Y. p. 32, 1977.
21.
Wagstaff DJ, Street JC. Ascorbic acid deficiency and induction of hepatic microsomal hydroxylative enzymes by organochlorine pesticides. Toxicol. Appl. Pharmacol. 19:10-19, 1971.
22.
Reid ME. Nutrient requirements of the guinea pigs. Nutrient requirements of laboratory animals number X. A report of the committee on Animal Nutrition. National Academy of Sciences. National Research Council. pp 11-23, 1962.
23.
Gabovich RD. Maistruk PN. On the therapeutic and prophylactic diet in the fluroine manufacturing industry. Vopr@sy Pitaniia 22:32-38, 1963.
24.
Varghese AJ, Land PC, Furrer R, Bruce WR. Non-volatile n-nitroso compounds in human feces. In: Environmental aspects of n-nitroso compounds. International Agency for Research on Cancer. IARC Scientific Publications. No. 19, 257-264, 1978.
25.
Chakraborty D, et al. Biochemical studies on polychlorinated biphenyl toxicity in rats: manipulation by vitamin C. Internat. J. Vit. Nutr. Res. 8(1):22-31, 1978.
26.
Calabrese EJ, Sorensen AJ. The health effects of PCBs with particular emphasis on human high risk groups. Rev. on Environ. Health. 2(4): 285-304, 1977.
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Environ. Health
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Blackstone S, Hurley RI, Hughes RE. Some interrelationship between Food vitamin C (L-ascorbic acid) and mercury in the guinea pig. -and Cosmetic Toxicology. 12:511-516, 1974.
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Conrad ME, Barton JC. Factors affecting the absorption and excretion of lead in the rat. Gasteroenterology 74~731-740, 1978.
29.
Basu TK. acid.
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