Recent advances in the toxicity of heavy metals—An overview

FUNDAMENTAL AND APPLIED TOXICOLOGY 1:348-352 (1981)

Recent Advances in the Toxicity of Heavy Metals - An O v e r v i e w GEORGE C. BECKING Environmental and Occupational Toxicology Division, Environmental Health Directorate, Health Protection Branch, Department of National Health and Welfare, Ottawa, Canada

GENERAL I N T R O D U C T I O N In the past, studies on the toxicity of heavy metals concentrated on the gross effects, using doses greatly exceeding the normal physiological defense processes. Due to greatly increased anthropogenic activity, people are continually exposed to low levels of heavy metals in their total environment. As toxicologists, we must be prepared to delineate the potential health hazards from such exposures. Only by careful study of the subtle effects of low concentrations of metals can we accomplish this task. In this paper, an attempt will be made to review some of the recent advances in the two following areas: (1) metals as mutagens a n d / o r carcinogens, and (2) neurotoxic and behavioral effects of heavy metals. It will be necessary to discuss briefly other areas, such as chemical speciation and metabolism in order to fully evaluate the advances made in these two major areas of concern. Also, it is hoped to show how most major advances have resulted from m u l t i d i s c i p l i n a r y approaches. A combination of such disciplines as genetic toxicology, psychology, as well as more classical toxicology, has resulted in the development of many new hypotheses and a better understanding of the toxic properties of metals and their mechanism of biological action. METALS AS MUTAGENS/CARCINOGENS Introduction N o t w i t h s t a n d i n g the many reviews on this subject (Furst and Haro, 1969; Furst, 1977, 1978; Sunderman, 1977; L~onard, 1979; Norseth, 1977), there is still much controversy over the actual occupational and environmental hazard posed by heavy metals. How far have we progressed? The various metallic elements previously reviewed for carcinogenic potential are shown in Table 1. Of those shown, only beryllium, cadmium, cobalt, iron, lead and nickel have been found to induce cancer in various species of experimental animals after injection, implanation or inhalation of specific chemical species. In general, oral administration of metallic compounds has failed to produce tumors. We then must rely on epidemiological ifivestigations, imprecise as they are, to delineate the potential of metals to cause cancer in man. A recent workshop on the role of metals in carcinogenesis evaluated the available data and concluded that those metals shown in Table 2 can contribute to human cancer (Belman, 1981). You will note that the ubiquitous environmental pollutant, lead, an animal carcinogen at extremely high oral doses (Van Esch et al., 1962; Van Esch and Kroes, 1969), has not been included; in agreement with the conclusions reached in a recent review by Moore and Meredith (1979). In summary, insufficient observations on man exposed

to lead only have been reported to enable a definite conclusion to be reached regarding the carcinogenicity of lead in man - an unfortunate situation. Given the equivocal results of both mutagenicity assays on lead (Gerber eta/., 1980) and occupational epidemiological investigations, we have an extremely important area ripe for future research. As will be seen later, perhaps in vitro mutagenicity studies will be of value in any future investigations of the carcinogenicity of lead. In the area of metals as carcinogens, with one major exception, we are nowhere near answering such questions as: (1) are metallic elements and their compounds primarily carcinogens? (2) are such elements and substances co-carcinogens? (3) do inorganic substances pose a mutagenic risk to man? - an extremely important question; (4) what mechanisms are involved? That is, what is the role of chemical speciation a n d / o r metabolism of the element in the production of mutations or cancer?; and (5) are the metallic elements themselves carcinogenic or only specific chemical species? Use o f in vitro tests in the study o f metallic carcinogens During the last decade, many in vitro systems have been developed to study the interaction of chemicals with cellular components, including the genetic apparatus and the enzymes associated with replication. In vitro models widely used in the study of metallic compounds are summarized in Table 3. Some of the problems which can be addressed by such in vitro systems are indicated in Table 4. As with organic chemicals, batteries of short term tests should be useful in prescreening for carcinogenic and mutagenic metals. No one test will suffice. In fact, metals appear to behave similarly to known organic carcinogens in in vitro transformation assays, but are frequently negative in bacterial reverse mutation assays. The simplicity of subcellular or unicellular in vitro assays makes these systems useful in delineating mechanisms of genotoxicity as well as interactive effects w i t h i n mixtures of metals. TABLE 1 Metallic Elements Previously Reviewed for Carcinogenic Potential Ni Cr Cd Be Pb As

Zn Co Fe AI Cu Se

Hg Ti Sn Ag Mn

Copyright 1981, Society of Toxicology

348

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September/October, 1981

SYMPOSIUM ON PERSPECTIVES IN METAL TOXICITY TABLE 2 M e t a l l i c E l e m e n t s C a r c i n o g e n i c to Man Date and Type of Evidence Available Element

Case Reports

Epidemiology Studies

Animal Studies

As

1888

1948

(1979)*

Cr

1930"s

1948

(1958) 1970

Ni

1932

1958

1959

Be

1980

(1946) 1953

Cd

(1965) 1980

(1962)

*Dates in parenthesis refer to the time suggestive evidence for carcinogenicity was available.

Arsenic When evaluating the risk to man from arsenic, we are faced w i t h an unusual toxicological data package. That is, carcinogenicity data on arsenic reviewed by L~onard (1979) and L~onard and Lauwerys (1980) failed to identify arsenic as an animal carcinogen, but epidemiological data do implicate various arsenicals |particularlyltrivalent arsenic) as human carcinogens. However, available data cannot differentiate between arsenic as a primary carcinogen, a co-carcinogen or a promoter. L6onard and Lauwerys (1980) have concluded that arsenic is probably a huma n co-carcinogen. After 100 years of study, the mechanism by which arsenic causes cancer remains unknown, as does the actual chemical species leading to positive carcinogenic findings in humans and negative data in animal bioassays.

There is no doubt from the data shown in Table 5 that various arsenicals can cause genetic damage or alter DNA repair. Positive results were obtained both in vitro and in vivo, but have we studied the appropriate chemical species in an attempt to account for the difference in carcinogenicity in man and animals. Also, are there different genetic risks to man from the various arsenicals in his environment? Can metabolic studies in concert with short term tests help answer such questions? The extensive metabolic data on arsenic from several laboratories (Crecelius, 1977; Charbonneau et al., 1978, 1979 and 1980; and Odanaka et al., 1980) are summarized briefly in Table 6. Such data indicate man is the only species which excretes monomethylarsonic acid as a major metabolite. It is tempting to speculate that such results explain the different biological responses to arsenicals, but results of short term assays on many species of arsenic (Table 5) indicate a more complex explanation is required. Perhaps short term tests will indeed provide answers to this perplexing toxicological problem. Cadmium With cadmium, we are also faced with controversial results from epidemiological studies on human carcinogenicity, as well as equivocal results using in vitro assays to study mutagenic potential. At a recent workshop (Belman, 1981 ), it was concluded that cadmium led to the development of prostatic cancer in exposed Workers. Such a conclusion seems tenuous, based on the Fundamental and Applied Toxicology

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number of workers studied, the unknown cadmium exposures, possible exposure to other .metals, and the smoking habits of the subjects. A previous review by L6onard (1979) does not agree with the conclusion that cadmium is a human carcinogen. The results from various short term assays on cadmium shown in Table 7 tend to support the conclusion that cadmium is non-mutagenic, but one must explain the cell transformation activity and minimal effects on DNA repair, particularly regarding potential carcinogenicity. The possibility that cadmium is a co-m utagen (carcinogen) has recently received support from studies by Rubin and Kroll (1981 ). At non-toxic levels (approximately 2 X 10 -s) cadmium was non-mutagenic to TA98 Salmonella, but did increase 3-4 fold the mutagenicityof hycanthone, a known direct-acting mutagen. Studies with metabolic activation were difficult, since cadmium inhibited the activity of the metabolic enzymes. Such tests should be expanded to mammalian systems to attempt to determine the carcinogenic risk to man from cadmium. The major source of intake of cadmium in the general population is in food. A high percentage of this metal may be as the cadmium-metallothionein complex. Cherian (1979) has shown that this complex behaves differently from the inorganic metal and is more nephrotoxic, but no short term mutagenicity tests have been carried out on this cadmium species. It is therefore not clear what role cadmium-metallothionein plays in the mutagenicity of this metal in vivo. Chromium There is no doubt that chromium is a mammalian (human) carcinogen (L6onard, 1979) but until recently the role of speciation in the mutagenic/carcinogenic process was not.clear, although the hexavalent form was strongly implicated. Of the metals implicated in causing human cancer, chromium is one of the most widely studied in various in vitro systems (DiPaolo and Casto, 1979; Macrae et al., 1979; Nestmann et al., 1979; Whiting et al., 1979; Douglas et al., 1980). TABLE 3 in vitro M o d e l s for S t u d i e s of M e t a l l i c C a r c i n o g e n s

1.

Bacterial Mutagenesis

2.

Mammalian Cell Mutagenesis

3.

in vitro Cell Transformation

(a) (b)

Direct Chemical Transformation Viral-Chemical Interactions

4.

Cell Free Systems

5.

in vitro Cytogenetics

TABLE 4 Role of in vitro Assays in the S t u d y of M e t a l l i c Mutagens/Carcinogens 1.

Identification of Metallic Mutagens/Carcinogens (including mixtures)

2.

Analysis of Mechanisms of Metal Mutagenesis-Carcinogenesis

3.

Study of Interactions (a) Synergistic (b) Antagonistic (c) Cocarcinogenesis

4.

Determination of Agents Requiring Promotion

349

A summary of the pertinent results from in vitro studies on chromium is presented in Table 8. Data in the literature implicate hexavalent chromium as the active mutagenic/carcinogenic species, but two pieces of information must be considered before accepting this conclusion. Only Crs§ was active in bacterial (Nestmann, 1979) assays, but Petrilli and DeFIora (1978) found chromite (Cr 3~) very active if oxidized with permangate. Chromite cannot enter cell membranes, but it is extremely reactive towards DNA bases in isolated nucleic acids. Recently, Jennette (1979, 1981 ) reported that chromate is reduced in vivo in the presence of NADPH and microsomal enzymes. The trivalent chromium now inside the cell can react with critical receptors, including DNA, thus inducing genotoxic effects. Based on these data, and those from Table 8, a hypothetical mechanism for chromium carcinogenicity is presented in Table 9. Th us, with chromium, evidence from in vitro tests suggests that direct mutagenic initiation of somatic cells via DNA interactions is involved in chromium carcinogenesis. One metal has thus been shown as a direct acting carcinogen and a probable mechanism worked out largely by the use of short term tests. N E U R O T O X I C I T Y A N D B E H A V/ORAL EFFECTS OF M E T A L S Introduction Neurotoxic and behavioral effects may be sensitive indicators of adverse effects of metals. It is essential to design tests to detect such effects so that controls can be implemented before such reactions become permanent. To do this, toxicology must incorporate techniques from psychology into its armamentarium, but not forget other biological data when interpreting the meaning of these newtesting methodologies. This overview will discuss only the effects of metals on behavior. There is insufficient time to discuss neurotoxicity in any detail.

Those interested in recent advances in methodologies in behavioral toxicology should consult papers by Tilson and Cabe (1978), Vorhees et al. (1979) and Rice (1979). In the time available, it is impossible even to discuss fully the complicated field of behavioral toxicology of metals. Therefore, only examples of advances in the behavioral toxicology of lead TABLE 5 M u t a g e n i c i t y of A r s e n i c a l s *

Test (a) Microbial Rec-Assay

Chemical Species

Results

A s 3"

.-}-

As-organo

Reversion Assay

As~"

+

A s s* As-organo

(b)

Mammalian in vitro Cytogenics

in vivo Cytogenics

A s 3"

+

As 5" As-organo

+ +

Occupational Medical

+ +

*As 3" inhibits post replicative repair and Ass. transforms Syrian hamster embryo cells in vitro.

350

TABLE 6 Major Metabolites Following Ingestion of Inorganic Arsenic Chemical Species Excreted*

Man Monkey Dog Pig Rat Hamster

Inorganic

MMA

DMAA

+ + + + + +

+ trace trace

+ + + + + +

*Inorganic - total arsenite and arsenate MMA - monomethylarsonic acid DMMA - dimethylarsinic acid

and aluminum will be discussed. However, readers should be aware of recent advances in studies on the effects of methylmercury on behavior (Weiss, 1979; Bornhausen et al., 1980; Reuhl and Chang, 1979; and Joiner and Hupp, 1978). Lead Controversy still surrounds the conclusion of Byers and Lord (1943) that one aftermath of a cute lead poisoning was a deficit in behavior and learning ability. In the last fewyears, epidemiology has been forced far beyond its conventional limits, attempting to show an association between asymptomatic lead exposures and behavioral patterns. Experimental toxicologists have made some advances in this area of behavioral toxicology by utilizing various animal models and techniques of psychological testing. However, more effort must be expended to verify the relevance of any noted behavioral changes in animals to man. Recent studies by Rice et aL (1979) and Bushnell and Bowman (1979) in monkeys, and Cory-Slechta and Thompson (1979) in rats, have shown effects on behavior and learning from chronic exposure to relatively low levels of lead. The experiments of Cory-Slechta and Thompson (1979) found rats receiving between 12 and 40 mg/kg of lead had altered behavior patterns, as measured by fixed interval performance. Such a dosing regimen would result in brain lead levels of between 40 - 1080 ng Pb/g wet tissue, compared to control values of 14-26 ng/g. Studies by Rice et al. (1979a,b) and Bushnell and Bowman (1979) are of similar design. Rice and coworkers were more interested in activity and learning behavior, whereas Bushnell and Bowman studied social behavior in monkeys dosed chronically with lead immediately after birth. Bushnell and Bowman (1979) used dose levels w h i c h resulted in blood leads of approximately 80/~g/100 mL at the high dose level and 5 0 / ~ g / l O 0 mL in the low dose level. At these blood lead levels, monkeys exhibited disruptions in social behavior such as suppressed play habits and increased social clinging when compared to control animals. In the experiments by Rice et al, (1979a,b), monkeys were dosed by gavage (500 ~g/kg body weight) from day 1 of life for periods up to 3 years. No overt signs of lead toxicity were observed and blood leads peaked at 50-60/~g/100 mL at 200 days of age and declined to a stable level of 20-30/~g/100 mL prior to testing. This dosing regimen and resulting blood lead Fundam. Appl. Toxlcol. (1)

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SYMPOSIUM ON PERSPECTIVES IN METAL TOXICITY TABLE 7 R e s u l t s f r o m in vivo A s s a y s o n C a d m i u m 1.

Transformation of Syrian Hamster Embryo Cells

2.

Chromosome Damage in Plants

3.

Negative in Frameshift or Base Pair Mutational Assays

4.

Failure to Produce Sex-Linked Recessive Lethals in Drosophila

5.

Indications of Effect on DNA Repair

6.

Equivocalin vitro Cytogenetics

7.

Failure to Induce Chromosome Aberrations in vivo

CONCL UDING REMARKS

*Tests on various inorganic salts - not on orga no-cadmium complexes.

TABLE 8 C h r o m i u m - R e s u l t s f r o m in vitro A s s a y s

1.

Transformation of Syrian Hamster Embryo Cells

2.

Mutagenic (Cr ~') in Bacteria and Mammalian Cells in Culture (Cytogenetics)

3.

Chromite (Cr 3") Active in Cell-Free Genetic Tests - Not Absorbed Through Membranes

4.

C h r o m a t e Induces DNA Damage and Unscheduled DNA Synthesis in Mammalian Cells

5.

Chromate Induces Micronuclei in Mouse Bone Marrow

levels are not out of line with present findings in many urban children. At these blood lead levels, little change in locomotor activity was noted, but social behavior patterns similar to those of Bushnell and Bowman (1979) were noted. If indeed changes in behavioral patterns of monkeys are directly applicable to children, these workers have developed a powerful tool for the study of minimal toxic effects of metals. Aluminum

Aluminum, one of the most widely distributed metals, has not been extensively studied for adverse biological effects. Until recently, the gastrointestinal tract was considered a sufficient barrier to prevent overt toxicity from this ubiquitous element. This seems a reasonable hypothesis, since the body burden in man is less than 30 rag, even though exposure to reasonably high levels of aluminum is relatively constant. Studies by Bowdler et aL (1979), have raised many questions concerning the neurotoxicity of aluminum and its effects on human behavior. The conclusions reached by the authors regarding cause and effect relationships between aluminum exposure and behavioral effects in humans are not fully supported by the data presented. In fact, all the blood levels shown in Table 10 are considered to be w i t h i n the normal non-toxic range, and one must question whether the differences noted in the behavioral tests were simplydue to an inherent error of the tests and the normal variability between subjects. Notwithstanding the above criticisms, Bowdler et al. (1979) have raised certain questionswhich need further study. This is particularlytrue, given the recent summary by McLachlan and DeBoni (1980) on the role of aluminum in human brain disease. This area is in need of good multidisciplinary studies to either delineate the true adverse effects of aluminum, Fundamental and Applied Toxicology

or to obtain data sufficient to conclude that there is no causal relationship between normal aluminum exposures and human disease.

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In preparing a short overview on such a wide-ranging subject, I am sure I have omitted favorite areas of many people. For example, I have not attempted to review the advances in the area of metals a nd the immune system. Some recent papers by Blakely et al. (1980), Graham et al. (1978) and Lawrence (1981) are examples of advances made in this area. Also, I have not attempted to review recent advances in the teratogenic effects of metals. Reviews on the teratogenicity of lead, arsenic and cadmium have recently been published (L~onard and Lauwerys, 1980; Gerber et al., 1980; Degroeve, 1981 ). It is my opinion that toxicology has just started to unravel the complex field of metal toxicology. Only by continual integration of many disciplines will progress be made in further delineating the many possible complex interactions of metals and the full role of heavy metals in health and disease. REFERENCES Belman, S. (1981). Editor in Chief, Proceedings of W o r k s h o p o n the role of metals in carcinogenesis. In: Environ. Health Perspect. 38 (In Press). Blakley, B.R., Sisodia, C.S. and M u k k u r , T.K. (1980). T h e effect of m e t h y l m e r c u r y , t e t r a e t h y l lead, and sodium a r s e n i t e on the h o r m o n a l i m m u n e response in mice. ToxicoL AppL Pharmacol. 52:245-254. B o r n h a u s e n , M., Musch, H.R. a n d G r e i m , H. (1980). O p e r a n t b e h a v i o r p e r f o r m a n c e c h a n g e s in rats a f t e r p r e n a t a l m e t h y l m e r c u r y exposure. ToxicoL AppL Pharmacol. 56:305-310. Bowdler, N.C., Beasley, D.G., Fritze, E.C., G o u l e t t e , A.M., H a t t o n ,

TABLE 9 H y p o t h e s i s f o r M e c h a n i s m of C h r o m i u m C a r c i n o g e n e s i s 1.

Animal Cancer Studies Implicate Cr ~"

2.

Mutagenic Activity of Various Chromium Compounds Markedly Dependent on Oxidation State

3.

Results Explicable in Terms of the Uptake-Reduction Model of Jennette (1979, 1981)

4.

Cr 3" Binds Directly to Nucleotide Bases in DNA - C h r o m i u m Probably a Mutagenic Initiator

TABLE 1 0 Aluminum Intake and Behavioral and Neurological Functions in Humans Test Scores (-t- SE) Test Digit Symbol Serial Sevens - Pauses Serial Sevens - Errors Flicker Frequency Trails B

No. Subjects 21 14 16, 15 24, 25 20

AI Intake" Low 15.1 0.4 1.4 34.8 107.8

-{- 0.6 -t- 0.2 + 0.3 -t- 0.8 -I- 8.4

High 13.5 2.6 2.5 37.2 125.0

-t- 0.6 -I- 0.8 + 0.7 -4- 0.6 • 9.0

"Aluminum intake inferred from serum levels: High - 504 nglmL; Low - 387 nglmL (Bowdler et aL, 1979) P<0.05. 351

I.D., Hession, l., Ostman, D.L., Rugg, D.J. and Schmittdiel, C.J. (1979). Behavioral effects of aluminum ingestion on animal and human subjects. Pharmacol. Biochem. Behav. 10:505-512.

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further studies. In: Trace Metals Exposure and Health Effects (E. DiFerrante, ed.) pp. 199-216. Pergamon Press, Oxford. L~onard, A. and Lauwerys, R.R. (1980). Carcinogenicity, teratogenicity and mutagenicity of arsenic. Mutation Res. 75:49-62. Macrae, W.D., Shiting, R.F. and Stitch, H.F. (1979). Sister chromatid exchanges induced in cultured mammalian cells by chromate. Chem.-Biol. Interact. 26:281-286. Moore, M.R. and Meredith, P.A. (1979). The carcinogenicity of lead. Arch. ToxicoL 42:87-94. Nestmann, E.R., Matula, T.I., Douglas, G.R., Bora, K.C. and Kowbel, D.J. (1979). Detection of the mutagenic activity of lead chromate using a battery of microbial tests. Mutation Res. 66:357-365. Norseth, T. (1977). Industrial viewpoints on cancer caused by metals as a n occupational disease. In: Origins of Human Cancer. Vol. 4 (A.H.H. Hiatt, J.D. Watson and J.A. Winsten, eds.) pp. 159-167, Cold Spring Harbor Laboratory. Odanaka, Y., Matano, D. and Goto, S. (1980). Biomethylation of inorganic arsenic by the rat and some laboratory animals. Bull. Environ. Contain. Toxicol. 24:452-459. Petrilli, lr.L and DeFlora, S. (1978). Oxidation of inactive trivalent chromium to the mutagenic hexavalent form. Mutation Res. 58:167-173. Reuhl, K.R. and Chang, L.W. (1979). Effects of methylmercury on the development of the nervous system: A review. Neurotoxicolog.l' 1:21-55. Rice, D.C. (1979). Operant conditioning of infant monkeys (Macaca fascicularis) for toxicity testing. Neurobehav. ToxicoL 1, Suppl. 1:85-92. Rice, D.C., Gilbert, S.G. and Willes, R.F. (1979a). Neonatal lowlevel lead exposure in monkeys: Locomotor activity, schedulecontrolled behavior, and the effects of amphetamine. ToxicoL Appl. Pharmacol. 51:503-513.

Rice, D.C. and Willes, R.F. (1979b). Neonatal low-level lead exposure in monkeys (Macacafascicularis): Effect on t w o - c h o i c e non-spatial form discrimination. J. Environ. Pathol. ToxicoL 2:1195-1203. Rubin, R.I. and Kroll, R. (1981). Toxicity, mutagenesis, and comutagenesis of cadmium (Cd) in Salmonella mutants TA98 and TA100. The Toxicologist 1:25 (Abstract 92). Sunderman, Iz.W., lr. (1978). Carcinogenic effects of metals. Fed. Proc. 37:40-46. Sunderman, lr.W., Jr. (1977). Metal carcinogenesis. In: Advances in Modern Toxicology. (R.A. Goyer and M.A. Mehlmann, eds.) Vol. 2, pp. 257-295, John Wiley and Sons, New York. Tilson, H.A. and Cabe, P.A. (1978). Strategy for the assessment of neurobehavioral consequences of environmental factors. Environ. tleahh Perspect. 26:287-299. Van Esch, G.I. and Kroes, R. (1969). The induction of renal tumors by feeding basic lead acetate to mice and hamsters Br. J. Cancer 23:765-771. Van Esch, G.J., Van Genderen, H. and Vink, H.H. (1962). The induction of renal tumors by feeding basic lead acetate to rats. Br. J. Cancer 16:289-297. Vorhees, C.V., Butcher, R.E., Brunner, R.L. and Sobotka, T.J. (1979). A developmental test battery for neurobehavioral toxicity in rats: A preliminary analysis using monosodium glutamate, calcium carrageenan and hydroxyurea. ToxicoL AppL PharmacoL 50:267-282. Weiss, B. (1979). Behavior as a sentry of metal toxicity. In: Trace Metals Exposure and ttealth Effects (E. Diferrante, ed.) pp. 185-198. Pergamon Press, Oxford. Whiling, R.F., Stich, H.F. and Koropatnick, D.I. (1979). DNA damage and DNA repair in cultured human cells exposed to chromate. Chem.-BioL Interact. 26:267-280.

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