Mutagenicity and disposition of chromium

The Science of the Total Environment, 86 (1989) 131-148 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

131

MUTAGENICITY A N D DISPOSITION OF CHROMIUM*

CHARLOTTE M. WITMER** and HYOUNG-SOOK PARK

Rutgers University Graduate Program in Toxicology, Rutgers University, The State University of New Jersey, New Brunswick, N J 08903 (U.S.A.) SAUL I. SHUPACK

Department of Chemistry, Villanova University, Villanova, PA 19085 (U.S.A.)

ABSTRACT Hexavalent chromium was administered to rats at doses of 20-240 #mol kg-1 for several periods of time, from 2 to 14 days. Lung, liver and blood contained the highest amounts of chromium, as detected by atomic absorption or by ICP, 24 h after cessation of treatment. A maximum of 40% of the dose was recoverable in organs along with feces and urine at this same time period, and chromium in soil (5.6% Cr) was absorbed better than equimolar amounts of the hexavalent chromates of calcium or sodium. The contaminated chromium-containing soil was found to have 30-35% of the chromium in the hexavalent state. The mutagenicity of chromium as tested in the bacterial strain of Salmonella typhimurium (strain TA 104) was decreased when tested without metabolic activation with the addition of leachate (of inexact analysis) from a waste site. When studied by alkaline elution, chromium (5-20#M) caused single strand breaks as well as DNAprotein crosslinks in A549 lung cells, while with L1210 mouse leukemia cells, only DNA-protein crosslinks were found. Chromium(III) compounds caused no damage to DNA.

INTRODUCTION

Chromium, in high concentrations, has been found in specific sites in the state of New Jersey as a result of dumping of chromium-containing waste several decades ago. The sites have become of public concern because of their proximity to populated areas and the resultant possibilities of children using these sites as play areas and ingesting some of the dirt. Chromium has also been found in drinking water from leachate sites and it is realized t hat there is also the possibility of ingestion of some amounts of chromium by the general population. As a result of this awareness, this study was originated to determine the bioavailability of chromium from soil from a specific site. In parallel with this, we studied the bioavailability of chromium from the pure chromium salts as well as f r o m a mixture of the calcium salt and the chromiumcontaining soil or slag. This is still in progress and therefore some of the studies which are an integral part of the project are not reported on here. *Parts of this work were presented at the 28th meeting of the Society of Toxicology, Atlanta, GA, March 1989. **Author to whom correspondence should be addresed. 0048-9697/89/$03.50

© 1989 Elsevier Science Publishers B.V.

132

Another part of this report includes some work in our laboratory to determine the different effects of chromium compounds on DNA from cells in culture from different sources. This work was carried out using A549 human lung carcinoma cells which have the properties of Type II alveolar epithelial cells, and with L1210 mouse leukemia cells. We are particularly interested in specific effects of chromium on human lung cells because of the epidemiological studies which indicate the incidence of lung cancer after exposure to chromium compounds (Langard, 1982; EPA, 1984). We have also studied the mutagenicity of chromium in bacterial systems to investigate possible synergistic or a t t e n u a t i n g effects with simultaneous exposure to other compounds in mixtures. This has been used by our group as a way to study the effects of mixtures. METHODS

For the bioavailability studies we have used male Spraque-Dawley rats (80-100g) as the experimental animals. The animals were purchased from Taconic Farms (Germantown, NY) and only heal t hy animals were used. They were acclimated to our vivarium for several days before the start of any treatment, and during the t r e a t m ent periods they were kept i n the animal quarters with a light/dark cycle of 12 h each. All other t r e a t m e n t followed the guidelines of the University animal committee. All chromium salts were the purest available. The chromium salts and soil were administered orally to the rats and the animals were sacrificed at the times designated for each experiment. For the detection of chromium we originally used the atomic absorption (AA) technique (with a graphite furnace), but the more recent work, as indicated, was carried out using the Baird Inductively Coupled Plasma (ICP) instrument, in which the element is detected by atomic emission and the atomization cell is an argon plasma. The ICP instrument gave us the most sensitive results, although for the lower concentrations of chromium, the AA method was very acceptable. Neither instrument differentiates between chromium in the different oxidation states. Samples were prepared for analysis by a modification of the acid procedure, the procedure recommended by the National Bureau of Standards. Briefly, 0.1 g sample (of tissue or other source of chromium) was weighed into a 20 ml test tube and 1 ml of concentrated HNO3 was added slowly. After the brown fumes had subsided, 1 ml of 30% H2 O2 was added. After the ebullition and foaming had ceased, the solution was carefully warmed in a water bath to 60°C and kept at t hat temperature for 16 h or more. When the solution was clear, the contents were transferred to a 25ml volumetric flask and the volume made up to 25 ml with double distilled water. Aliquots were then assayed for chromium using either of the two instruments, as above. If necessary, the solutions were filtered prior to analysis. Standards of chromium (Sigma Chemical Company) were used to standardize each instrument. In the cell culture studies the effects of chromium on DNA in these cells were measured by the t e c h n i q u e of alkaline elution, using a modification of the

133

method of Kohn et al. (1981). In these studies, DNA was labeled in exponentially growing cells with ltC or 3H thymidine for 1 or 2 cell generations. (Reference or internal standard cells were labeled with 3H and sample cells labeled with ltC.) The cells were then treated with the chromium compound for 2-3 h and then applied to the filter. On the filter, the cells were lysed and the double-stranded DNA converted to single-stranded DNA (SS) by an alkaline solution, pill2.1. The SS DNA is able to pass through the filters. This technique measures the elution rate of the SS DNA by detection of the radioactivity in the eluted samples and then determining the fraction of label left on the filter. The flow rate is controlled by a peristaltic pump and the rate of flow for our experiments was maintained at 0.035ml min 1. The fractions were collected at 90 min intervals and the radioactivity in the fractions counted in a Tr aco r Analytic, Mark III counter. Variations of technique such as the use of different filters, X-ray t r e a t m e n t and proteinase K t reat m ent allow the determination of DNA breaks and DNA-protein cross-links. The resulting 14C and 3H cpm of each eluted fraction can be calculated as the percentage of the total cpm. The percentages of sample DNA and of reference DNA remaining on the filter are then plotted on a log-log scale graph. 1.0 A

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Fig. 1. The effects of K2CrO 4 on DNA single-strand breaks in A549 h u m a n l u n g carcinoma cells. Cells were exposed to 0, 5, 10, and 2 0 ~ M K2Cr207 for 3 h at 37°C. Alkaline elution assays were carried out without the use of X-rays and with t r e a t m e n t by proteinase K. Method is described in the text.

134 RESULTS

Alkaline elution studies Figure 1 shows the effects of hexavalent potassium dichromate on DNA SS breaks in A549 cells. The cells were exposed to 0, 5, 10, and 20 #M dichromate for 3 h. Proteinase K was added to the lysing solution to eliminate protein. The SS breaks in the DNA allow the DNA to pass t hrough the filter more rapidly th an the unbroken DNA. There is a dose-related response between the concentration of hexavalent chromium to which the cells were exposed and the numbers of SS breaks. Figure 2 shows the effect of hexavalent chromium (potassium dichromate) on DNA-protein cross linking in A549 cells. The exponentially growing cells were exposed to 0, 10 and 20/~M dichromate for 3 h at 37°C. All cells were then treated with y-ray to produce a standard amount of SS breaks. D N A - pr ot e i n cross-links cause the DNA to pass t hrough the filter more slowly t h a n control DNA (which, in this case, had been treated with 7-ray 1.0 ~i[,]:l "

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Fig. 3. T h e effects of N a 2CrO 4 on D N A a l k a l i n e e l u t i o n k i n e t i c s in L1210 cells. Cells were e x p o s e d to 5 ~ M Na2CrO4 for 2 h at 37°C. A l k a l i n e e l u t i o n a s s a y s were c a r r i e d o u t u n d e r t h e c o n d i t i o n s d e s i g n a t e d in t h e figure.

but not with chromium). As is shown, hexavalent chromium induces D N A protein crosslinks in a dose-response manner. Figure 3 shows the effects of hexavalent chromium as sodium chromate on DNA alkaline elution in L1210 cells. These cells were exposed to 5 pM hexavalent chromium for 2 h. No SS breaks were found in these cells, although DNA-protein crosslinks were present. To determine whether these cells were sensitive to the same types of damage on exposure to trivalent chromium compounds, A549 cells were exposed to CrC13"6H2 O for 3 h. By the addition of proteinase K to some samples and by irradiation, the studies assayed for both SS breaks and DNA-protein crosslinks. Figure 4 shows t hat neither of these types of damage were found following exposure of the A549 cells to chromium(III). Figure 5 shows the results of cell toxicity studies for A549 cells on exposure to hexavalent dichromate or trivalent chromium chloride. The cells were exposed to the specific compounds for 3 h and then incubated in a chromium-free medium for 4 days. On Day 5 the surviving cells were counted and the number expressed as the percentage of the control cells. From Fig. 4 we see that potassium

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dichromate has an ICs0 of about 10 #M and t hat chromium(III) has no overall toxicity for these cells. These studies indicate t hat hexavalent chromium induced DNA SS breaks and DNA-protein crosslinks in A549 cells, but only DNA-protein crosslinks in L1210 cells. Thus, the DNA lesions are different in different cells. The lack of DNA damage found in A549 cells treated with trivalent chromium supports previous indications t hat hexavalent chromium crosses the cell membrane while trivalent chromium is excluded from cells. A difference in the response of lung cells to chromium may contribute to the preferential development of lung tumors following exposure to chromium in the environment, along with the r e t ent i on of small chromium-containing particles in the alveoli.

Chromium absorption studies Our initial studies were carried out by oral administration to rats of chromium(VI) as the soluble sodium salt. The first studies were range finding to determine tissue distribution of the chromium. In later studies we adminis-

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Fig. 5. C y t o t o x i c i t y of A549 cells. Cells were added to 25 ml flasks c o n t a i n i n g 5 ml of m e d i u m on D a y 1 at a c o n c e n t r a t i o n of 1 × 10~ cells ml - t . O n D a y 2, t h e cells were exposed to K~CrO 4 or CrC13 for 3 h a n d t h e n i n c u b a t e d in drug-free m e d i u m for 4 m o r e days. T h e s u r v i v i n g cells were c o u n t e d on D a y 5, w i t h c o u n t s e x p r e s s e d as a p e r c e n t a g e of c o n t r o l cells.

tered the calcium salt also, as analysis of a soil sample from the specific site investigated showed a high percentage of calcium in this site (72-80%, by scanning electron microscope detection), perhaps indicating a limed soil. All studies were carried out by oral administration of the chromium material. In the first experiment the rats were dosed with 0, 20, 40 and 100 pmol kg-1 of hexavalent chromium (as Na2 CrO4" 4H2 O, dissolved in distilled water) for 7 days, in a volume of 0.25 ml/100 g body weight. Control animals received an equal volume of distilled water. In this experiment the rats were divided randomly into four groups of three rats each, and dosed daily between 9 and 10 a.m. with the chromium compounds. The rats were weighed and sacrificed by anesthetic and guillotine 24 h after the last treatment. The following organs were removed and weighed for analysis of chromium content: liver, spleen, lung, and kidney. A sample of blood was also taken from the abdominal aorta for chromium analysis. The liver was weighed and a small aliquot taken for analysis; all other organs were used as the whole organ. Immediately after weighing, the organs were placed in l ml of nitric acid prior to further treatment, as above. The analyses for this experiment were carried out with the atomic absorption instrument. Figure 6 shows the results of the chromium analyses as total chromium/organ. It can be seen that the liver and kidney contain the highest amounts of chromium. When the data are expressed as ttg Cr/g organ, the kidney is seen to contain the highest concentration of

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Fig. 6. Tissue distribution of chromium from Na2CrO 4, Rats were administered Na2CrO 4 orally, once daily, at levels of 0, 20, 40 and 100 #mol/kg body weight for 7 days. The animals were sacrificed on Day 8 by guillotine, and the organs immediately removed and analyzed for chromium content by atomic absorption. Chromium levels are per total organ.

chromium (not shown). Very little chromium is found in the tissues at the two lower doses of chromium, and it appears that the 100#mol kg -1 dose may overwhelm some transport mechanism or is crossing membranes by a different transport mechanism than the lower doses. However, even in these high dosed animals the total amount of chromium in the tissues represents only 1.7% of the amount administered in the previous 24 h. Thus there appears to be no appreciable storage of chromium in these organs, at this dose. It is possible that other organs contain a large amount of the ingested chromium or that the chromium is not absorbed to any greater extent than indicated by this low percentage. In the next experiment, the results of which are shown in Fig. 7, 120/~mol Cr/kg was administered orally to four groups of rats (three per group) for 7 days. These rats received the chromium from different sources, as follows: Na2CrO4, CaCrO4, soil containing the same amount of chromium (as determined by previous analysis using the ICP method), and a mixture of the soil and the CaCrO4 salt. This figure shows that whatever the source of

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Fig. 7. Tissue levels of chromium. Rats were administered chromium at a dose of 120 ~mol kg-1 for 7 days in the form of the two salts, Na2CrO 4 and CaCrO4, as well as in soil and in a mixture of soil and the calcium salt. They were sacrificed 24 h after the last treatment and the organs taken and analyzed for chromium using the ICP method. Chromium levels are per gram tissue.

chromium, the kidney again contains a higher concentration (ng/g tissue) thail does the liver. Also, the chromium concentration from soil (PAC) in the kidney is high. Under these conditions the content of the chromium from the sodium salt is higher than the content from the other sources of chromium. The levels of chromium from the administration of the sodium salt are generally much higher than those from the calcium salt in all tissues. The control values are unusually high in this experiment for an unknown reason. In the next experiment, the sources of chromium were the same but the dose levels were higher, 240/~mol Cr/kg, and the compounds were given for 14 days. There were six rats in each group of this experiment. The total tissue levels of chromium are again seen to represent a small percentage of the chromium administered (Fig. 8). Thus, the expected accumulation did not occur in any tissues, with the higher levels of dosing and the longer period of time. These results are given as nanograms per gram tissue, but the total tissue values

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Fig. 8. Tissue levels of chromium. Rats were administered the several salts of chromium, as designated in the figure, for 14 days at a level of 240 #mol Cr/kg body weight. They were sacrificed on Day 15 by ether anesthesia and by bleeding from the abdominal aorta. Tissues were analyzed for chromium content using the ICP method. Chromium levels are per gram tissue.

indicate that either very little of the chromium was absorbed or that it was immediately excreted. It is also apparent that, under these conditions of higher administration doses, chromium from any samples containing soil, either alone or as a mixture with the calcium salt, is higher in most tissues than is the chromium from either of the two chromate salts. To determine whether a substantial amount of the chromium is immediately excreted, the animals were placed in metabolic cages and the chromium content of urine and feces was determined following ingestion of the same hexavalent chromium. The protocol for the experiment to determine chromium excretion was as follows:

141

The groups were designated as: Group A - - control group; the rats were dosed with vehicle (deionized, distilled water; DD water) Group B - - CaCrO4 group; the chromate salt was made into a suspension in DD water and given as a 0.5 ml/200 g volume dose Group C - - Pacific Ave. soil group. The ground up soil was made into a suspension in DD water and given as 0.5ml/200 g. The amount of chromium given to Groups B and C was 240 #mol/kg body weight. The chromium was administered orally as a suspension in distilled water, for 7 days, once daily between 9 and 10. a.m. However, the urine and feces were collected only on the first 2 days and on Days 7 and 8. The analyses for Days 7 and 8 are not yet completed and thus are not reported here. Urine and feces were collected at 6, 12 and 24 h periods after the dosing. These urine and feces samples were then digested and prepared for analysis as described above. The chromium was determined using the inductively coupled plasma (ICP) method. The instrument was standardized using the chromium standards from Sigma. During this experiment, the animals were placed in metabolic cages and were kept there t h r o u g h o u t the time during which feces and urine were collected. The cages contained plastic (nalgene) partitions which separated the urine and feces immediately. In each case the urine volume was carefully determined and the feces weight was a wet weight after blotting the material. The rats were weighed daily and observed carefully for any abnormal behavior or unusual symptoms. The results of the excretion experiments are shown in Figs 9-11. These experiments indicated t hat chromium is not excreted to an appreciable extent in the urine and thus gastro-intestinal absorption appears to be low, in the order of 1-2%. Our early experiments support this finding, as does work of previous groups (Langard, 1982; Starich and Blincoe, 1983). A comparison of the urine levels following administration of the calcium and sodium salts at these relatively high levels of 240 #mol kg- 1 indicates t hat the chromium from the soil is absorbed to a greater extent t han t hat from the calcium salt, as seen in the greater excretion of chromium in the urine, i.e. 33.2 pg day i rat 1(range 17.8-47.1) in urine of Group C rats as opposed to 8.77 (range 5.7-11.7) in the urine of Group B animals. Since the excretion in the urine is a good indication of the amount absorbed and then excreted by the kidney (Anderson and Kozlovsky, 1985), this indicates a significant difference between the groups. The amount of chromium excreted in the feces of the rats which were fed the chromium from the soil was also much greater t han t hat excreted by the rats fed the calcium salt. However, the rats dosed with soil excreted 19% of the dose given within the previous 24 h, which is in some agreement with the data of Langard (1982), who found 17% excretion in feces for rats given 0.56mg of the sodium salt by the i.v. route. (Group B rats received an average of 1.8mg Cr day- 1and an absorption of 1.7% would indicate an amount of 0.032 mg reaching the circulation compared with the 0.56i.v. dose. There was no sodium salt

142

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administered in the excretion studies from which to draw a more direct comparison.) The 19% excretion implies that the chromium is excreted slowly, as at the time of measurement there was a total dose of 3.7 mg in comparison with the 1.8 mg dose in the 24 h just prior to sacrifice of the animals. The higher levels resulting from the chromate in soil may indicate that there is more biliary excretion of the chromium which has been absorbed, or it may indicate t h a t the chromate is bound to the soil and thus goes right through the gastrointestinal tract. Chromium is known to bind to clay soils. However, there is also the possibility t h a t the soil-fed rats have damaged intestinal walls so that more chromium passes through the membranes and also t h a t more is absorbed and perhaps recycled in the biliary tract. The greater excretion from the kidney may also indicate kidney damage.

143 C H R O M I U M E X C R E T I O N IN F E C E S / C O N T R O L 24

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A summary of these studies is as follows: CaCrO4-treated rats received an average dose of 1.8mg Cr day -1. Average total dose was 3.85mg Cr rat -1. At 24 h post-dosing, urinary excretion was 0.48% of the total dose. Chromium content in feces was 2.36% of the total dose. Soil-treated rats received an average dose of 1.83 mg Cr day-1. Average total dose was 3.73mg Cr rat -1. At 24 h post-dosing, urinary excretion was 1.80% of the total dose. Chromium content in feces was 19.1% of the total dose. One important question which arises from these studies is the question of the distribution of the chromium which has not been excreted and has not been found in the organs studied, by our group or by others. Previous data from other workers (Langard, 1982; EPA, 1984) support the findings that the lungs, liver and blood contain the major portion of the absorbed chromium and

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Fig. 10(a,b). Chromium excretion in urine (feces)/CaCrO4. Rats were administered chromium as CaCrO4 orally once daily (9-10 a.m.) for 2 days at a level of 240 ~mol kg-1 (0.5 ml/200 g). Urine and feces were collected 6, 12 and 24 h after each administration of the chromium salt. The urine and feces samples were digested as outlined in the text and chromium analyses were by the ICP method. Animal designations are as in Fig. 9,

indicate t h a t chromium is not bound to organs other t han those studied in our experiments, with the possible exception of the intestinal t r a c t and the bile. Some of the early reports in the literature were carried out before the analytical techniques for chromium detection were as sensitive as present methods, however, and may have to be reinterpreted. A major amount of the administered chromium still has not been accounted for, however.

Mutagenicity studies Initial studies were carried out to determine the effects of mixtures from several sources on the mutagenicity of chromium compounds using bacterial

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strains of S. typhimurium developed by Ames (Maron and Ames, 1983). The mutagenicity of Cr(VI) had been established with several strains of these bacteria (Lofroth, 1978; LaVelle and Witmer, 1984). These studies showed that a leachate sample which contained only low molecular weight fatty acids and other unidentified low molecular weight acids decreased the mutagenicity of hexavalent chromium compounds (Fig. 12). We have previously shown that chromium potentiates the mutagenicity of sodium azide (LaVelle and Witmer, 1986), but not that of methyl methanesulfonate. We are currently exploring other possible synergistic effects of chromium and other mixtures. The possible inhibitory effects of chromium on repair of DNA following specific types of damage are indicated by other studies. The question of whether chromium mutagenicity involves SOS repair is not settled, although our previous work clearly shows that the potentiation of mutagenicity takes place in a strain (TA 1535) which does not contain the pKM101 plasmid, which, if present, would increase the sensitivity to signals for SOS repair (Maron and Ames, 1983).

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Fig. ll(a,b). Chromium excretion in urine (feces)/soil. Rats were administered chromium-containing soil orally once daily (9-10 a.m.) for 2 days at a level of 240 ~mol kg-1 (0.5 ml/200 g). Urine and feces were collected 6, 12 and 24 h after each administration of the soil. The urine and feces samples were digested as outlined in the text and chromium analyses were by the ICP method. Animal designations are as in Fig. 9.

SUMMARY

It is apparent from these studies that the bioavailability of chromium salts depends on the form of the chromium, and on the degree of binding of the chromium to soil, in the case of chromium found in waste sites. Our preliminary studies have indicated that the chromium in the soil is present predominantly in the Cr(III) valence state. However, speciation of chromium has not yet been carefully examined in this soil and the possibility exists that the oxidation state has changed (and is still changing) on storage and exposure to oxygen. However, the two inorganic salts, both pure Cr(VI) compounds, differed in their absorption and tissue distribution and excretion patterns. Once

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hexavalent chromium is absorbed it is intracellularly reduced, apparently quantitatively, to Cr(III) (Wiegand et al., 1987), the putative toxic form which can react with DNA as a mutagen/carcinogen. Thus the absorption of the higher valence compounds has relevance for risk assessment. An understanding of the mechanism(s) of these putative toxic reactions is central to the use of data obtained from these and other studies for risk assessment. ACKNOWLEDGMENTS

This work was partially supported by a contract from the Department of Environmental Protection of the State of New Jersey and a grant from the Industry/University Cooperative Center for Research in Hazardous and Toxic Substances, at New Jersey Institute of Technology.

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Fig. 12. Effect of leachate on chromium mutagenicity in Ames strain 104. Mutagenicity was determined using S. typhimurium strain TA 104, developed by the Ames group (Maron and Ames, 1983), using a modification of the method outlined by Ames et al. (1975) REFERENCES Ames, B.N., J. McCann and E. Yamasaki, 1975. Methods for detecting carcinogens and mutagens with the salmonella/mammalian-microsome mutagenicity test. Mutat. Res., 31: 347-364. Anderson, R.A. and A. Kozlovsky, 1985. Chromium intake, absorption and excretion of subjects consuming self-selected diets. Am. J. Clin. Nutr., 41: 1177-1182. Environmental Protection Agency (EPA), 1984. Health Assessment Document for Chromium. EPA-600/8-83-014F. Environmental Criteria and Assessment Office, Research Triangle Park, NC, pp. 2-10. Kohn, K.W., R.A.G. Ewig, L.C. Erickson and L.A. Zwelling, 1981. Measurement of strand breaks and cross-links by alkaline elution. In: E.C. Friedberg and P.C. H a n a w a l t (Eds), DNA Repair: A Laboratory M a n u a l of Research Procedures. Marcel Dekker, Inc., New York. Langard, S., 1982. Absorption, transport and excretion of chromium in man and animals. In: S. Langard (Ed.), Biological and Environmental Aspects of Chromium. Elsevier Biomedical Press, Amsterdam. LaVelle, J.M. and C.M. Witmer, 1984. Chromium (VI) potentiates mutagenesis by sodium azide but not by methyl methansulfonate. Environ. Mutagen., 6: 311-320. Lofroth, G., 1978. The mutagenicity of hexavalent chromium is decreased by microsomal metabolism. Naturwissenschaften, 65: 207. Maron, D.M. and B. Ames, 1983. Revised methods for the salmonella mutagenicity test. Mutat. Res., 113: 173-215. Starich, G.H. and C. Blincoe, 1983. Dietary chromium - - Forms and availabilities. Sci. Total Environ. 28: 443-454. Wiegand, H.J., H. Ottenwalder and H.M. Bolt, 1987. Bioavailability and metabolism of hexavalent chromium compounds. Toxicol. Environ. Chem., 14: 163-275.