Environ. Sci. Technol. 1992,26,599-608
Determination of Mutagenicity in Sediments of the Aberjona Watershed Using Human Lymphoblast and Salmonella fyphimurium Mutation Assays John L. Durant’ and Harold F. Hemond
Parsons Laboratory for Water Resources and Aquatic Sciences, Department of Civil Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02 139 William
G.Thllly
Division of Toxicology, Center for Environmental Health Sciences. Whitaker College of Health Sciences and Technology, Cambridge, Massachusetts 02 139 Organic and inorganic chemical wastes from tanneries and insecticide and other industries have been released into the Aberjona watershed in eastern Massachusetts since before the midnineteenth century. In recent studies of the environmental fate of these waste chemicals, it has been shown that sediments in many parts of the watershed contain high concentrations of toxic metals and organic pollutants. We were concerned that exposure to these or other as yet uncharacterized pollutants in sediments might pose risks to human health. Thus, we collected 32 surface sediment samples downstream of major stream forks, from sediment deposition areas, and near known hazardous waste sites and tested their ability to mutate the bacterium Salmonella typhimurium and human lymphoblastoid cells. Dichloromethane-methanol extracts of 20 of the 32 samples were mutagenic to the bacteria, but only two were mutagenic to the human cells. This general lack of correlation between the two species’ responses is consistent with wide differences in sensitivitieswe have observed with pure chemical mutagens. We note with interest, however, that sediment mutagenicity was not significantly correlated with proximity to known hazardous waste sites, to present or former industrial sites, or with toxic metals concentrations. This may reflect either basin-wide redistribution of mutagenic chemicals or an origin not associated with identified industrial releases. The discovery of a potent mutagenic mixture in large quantity permits us to continue our study with the goal of identifying the primary mutagens, locating their sources, and evaluating the risks to human health.
I. Introduction Contamination of the aquatic environment is most often studied by measuring the concentrations of chemicals deemed a priori to pose the greatest risks to aquatic organisms and human health. This approach is based on the assumption that the identities of the most hazardous chemicals likely to be present in a contaminated area are known, and that an assessment of risk to humans can be made simply by analyzing water, sediment, and biota samples for those chemical species. A parallel approach which is becoming increasingly accepted is to test samples of “contaminated” materials for their ability to induce measurable biological change. In recent investigations of aquatic contamination, for example, samples have been tested in in vitro assays to assess mutagenicity or the ability to react with DNA. Using direct measurements of biological activity (e.g., mutagenicity) to assess the risks posed by aquatic contamination a t a specific site obviates the often fallacious assumption that the chemicals of greatest concern are those which have been previously classified as “pollutants” and are present in the highest concentrations. Biological assays can be used to estimate hazard by distinguishing biologically active from biologically inactive samples, detect compounds that may be 0013-936X192/0926-0599$03.00/0
dominant in terms of risk despite relatively low concentrations, and identify as yet unknown biologically active compounds. In studies where the genotoxic properties of aquatic contamination have been assessed, a number of in vitro assays have been employed. The most commonly used has been the Salmonella typhimurium histidine-dependence reversion assay or Ames test (1-19). In other tests, rat hepatocytes (unscheduled DNA synthesis) (9, 10, 13), Chinese hamster ovary cells (13),mouse embryo cells (20), nematodes (14), and Bacillis subtilis (17, 18) have been used. These studies have shown that mutagens are present in water (1+,20), fish tissue (7), and sediments (9-19) in many aquatic ecosystems. Noting the tendency for pollutants to accumulate in sediments, several researchers have made efforts to identify the principal classes of mutagens present in sediment samples. In isolating compounds responsible for S. typhimurium and rat hepatocyte genotoxicity in Black River (Ohio) sediments, West et al. (9) found that the mutagenic activity in n-pentane fractions of sediment extracts could be accounted for by polycyclic aromatic hydrocarbons with four to six aromatic rings. West et al. (10) also found that secondary and tertiary nitrogen heterocycles accounted for the mutagenic activity (S. typhimurium and rat hepatocyte) in chloroform extracts of Black River sediments. In contrast, Sat0 et al. found that four nitroarenes-2-nitrofluorene, 4,4’dinitrobiphenyl, 2,7-dinitrofluorene, and l-nitropyreneaccounted for the S. typhimurium mutagenicity in a benzene fraction of Suimon River (Gifu, Japan) sediments (11).
In these previous studies, the determination of mutagenicity was based on the results of testing in nonmammalian and/or “nonhumann mammalian (e.g., rat, hamster, mouse, etc.) cell assays. Although such in vitro tests are meant to serve as screening tools to assess the mutagenic and toxic properties of pure compounds and chemical mixtures to which humans could be exposed, these tests are not necessarily adequate predictors of biological activity in human tissue. To better approximate the human response to chemical mutagens, we have employed a human cell mutation assay (21). The assay involves human lymphoblast cells which have been induced to express a complement of human cytochrome P450 oxygenases and epoxide hydrolases known to be necessary for expressing biological activity of many environmental chemicals. In this paper we report on the first use of a human cell mutation assay in testing extracts of stream sediments. Our study area is the Aberjona watershed in eastern Massachusetts. The Aberjona watershed, a 25 square mile area located 10 miles north of Boston (Figure l), was once a major center for the production of finished leather and has long been prominent in the manufacture of chemicals, greenhouse products, electronic equipment, and other manufactured goods. Following the discovery of industrial
@ 1992 American Chemical Society
Environ. Sci. Technol., Vol. 26, No. 3, 1992 599
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Environ. Sci. Technoi., Vol. 26, No. 3, 1992
III. Results A. Mutagenicity and Toxicity in Human Lymphoblasts (MCL-3). Thirty-two sediment samples were tested in the human lymphoblast mutation assay (Figure 2). Of these, two were mutagenic: one from Little Brook (LBKS5500-900822) and one from Wedge Pond (WEPSW-900710). Additional samples collected at these sites (LBKS5500-910218 and WEPSW-910218) confirmed the initial results. The mean mutant fractions and relative survivals for each sample and concentration tested are shown in Table I. Dose-response curves for the Little Brook and Wedge Pond samples are shown in Figures 3
1.2 1t
0.2
1
0.2 1
--t
12 -
*
---it
OF-------
LBKS5500-900822 LBKS5500-910218 99% Confidence Limit
--t WEPSW-900710
WEPSW-910218
--ct
-
~
. . 99% Confidence
Limit I
0
50
100
'
150
'
200
'
250
'
3gO
'
Exposure Concentration (ugimL)
0
50
'
100
'
150
'
200
'
250
Exposure Concentration (ug/mL)
'
300
'
Figure 3. Mutagenicity in MCLB cells induced by 28-h exposure to extracts of Little Brook sediments LBKS5500-900822 and LBKS5500-910218. Each set of results is plotted as the mean mutant fraction f standard error for a single experiment performed in duplicate. For an exposure concentration to be mutagenic, the mutant fraction must be greater than the concurrent negative control (calculated using Dunnett's t test where p = 0.05), and it must exceed the 99 % upper confidence limit calculated from the historical negative control data base. Data points designated by asterisks fulfilled both statistical criteria.
Figure 4. Mutagenicity in MCL-3 cells induced by 28-h exposure to extracts of Wedge Pond sediments WEPSW-900710 and WEPSW910218. Each set of results is plotted as the mean mutant fraction f standard error for a single experiment performed in duplicate. For an exposure concentration to be mutagenic, the mutant fraction must be greater than the concurrent negative control (calculated using Dunnett's t test where p = 0.05), and it must exceed the 99 % upper confidence limit calculated from the historical negative control data base. Data points designated by asterisks fulfilled both statistical criteria.
and 4, respectively. All of the sediment samples which failed to meet both statistical criteria for mutagenicity were considered negative and not retested. Samples that were positive with respect to the concurrent control, but negative with respect to the historical negative control, were retested. All cultures exposed to test concentrations in excess of 100 pg/mL were turbid, suggesting that extract constituents were not soluble a t these concentrations. In 12 samples tested at concentrations greater than 100 pg/mL, survival fell below 20 % . Concurrent system blanks were neither mutagenic nor toxic in the assay (Table I). The first Little Brook sediment sample tested (LBKS5500-900822)exhibited mutagenicity at an exposure concentration of 100 pg/mL. At this dose the mean mua value which exceeded the tant fraction was 9.8 X concurrent negative control by 7 X lo+ and was greater than the 99% upper confidence limit for the historical negative controls. The second Little Brook sample (LBKS5500-910218) was mutagenic a t both 30 and 100 pg/mL. The mean mutant fraction a t 30 pg/mL was 8.4 x lo", while at 100 pg/mL the mean mutant fraction was 11.2 X These values satisfied both criteria for statistical significance. Neither Little Brook sample was mutagenic at 300 pg/mL, the highest concentration tested. At this concentration, the mean mutant fraction was 2.9 X for LBKS5500-900822 and 0.6 X for LBKS5500-910218. Such decreases in mutant fraction with increasing concentration may be associated with inducible DNA repair, detoxifying enzymes, or interactions among the chemical Constituents of the sample. In testing both samples, relative survival decreased with increasing concentration. At 300 pg/mL the relative survival was 69% for LBKS5500-900822 and 44% for LBKS5500910218.
The Wedge Pond sediment sample WEPSW-900710 was mutagenic at 100 pg/mL. At this concentration the mean mutant fraction was 7.5 X lo+. This was the only concentration at which WEPSW-900710 induced a mutagenic response. The second Wedge Pond sample tested (WEPSW-910218) was also mutagenic, but only at 30 pg/mL. At this concentration the mean mutant fraction was 8.0 X lo+. That the two samples were mutagenic a t different concentrations is not unusual. Such variability in responses between samples may be associated with random variations in the number of mutant cells surviving treatment, the number of mutant cells sampled after each growth-dilution cycle during the phenotypic expression period, and the number of mutant and nonmutant colonies actually observed (42). Both samples were quite toxic to the cells. Cells treated with WEPSW-900710 at a concentration of 300 pg/mL were all killed, and cells treated with 300 pg/mL of WEPSW-910218 had a relative survival of only 25%. To determine whether a sample which was mutagenic at high concentrations could also induce a mutagenic response at lower concentrations, a Wedge Pond sediment sample (WEPSW-900821) collected from the same site as the fist was tested at low concentrations (12.5-100 pg/mL) in a 72-h exposure assay, rather than the 28-h exposure assay. WEPSW-900821 induced a mutagenic response which was statistically significant when compared to the concurrent negative control but not when compared with the historical negative controls (Table I, Figure 5). WEPSW-900821 was also toxic to MCL-3 cells-cultures exposed a t 100 pg/mL had a relative survival of only 48%. A second long-term-low-dose experiment was performed with material from the WEPSW-910218 sample. The results for this sample confirmed the initial results: Environ. Sci. Technol., Vol. 26, No. 3, 1992
603
Table 11. Sediment Samples Tested for Mutagenicity in a S . typhimurium Forward Mutation Assay
-PMS" mutant fractiond concn'
(Xl06)
C 113 340 1020
7.3 f 1.5 9.5 f 1.7 10.3 f 1.7 11.6 f 2.3
relative survival'
+PMSb stat signiff CC HC
mutant fractiond (X106)
relative Survivale
stat signiff CC HC
ARS4400-900710 1.00
1.08 1.13 1.09
7.2 f 1.4 11.5 f 1.9
-
+
-
13.9 f 2.1 20.7 f 2.8
1.00 1.03 0.99 0.94
+ + +
9.1 f 1.3 14.1 f 1.7 19.9 f 2.3 22.8 f 3.2
1.00 1.02 0.86 0.52
+ +
5.7 f 1.4 12.6 f 2.3 17.6 f 3.1 23.8 f 4.0
1.00 0.94 0.75 0.66
+ + +
7.9 f 1.6 22.2 f 3.2 41.2 f 4.7 47.2 f 6.9
0.88 0.75 0.55
4.2 f 1.2 8.1 f 1.8 15.9 f 2.9 21.9 f 3.7
1.00 1.03 0.86 0.78
12.8 f 1.7 13.5 f 1.8 19.1 f 2.4 21.4 f 2.7
1.00 1.04 0.87 0.79
6.9 f 1.4 26.2 f 6.1 45.7 f 14.1 96.3 f 25.1
0.27 0.11 0.11
7.5 f 1.4 8.5 f 1.4 11.6 f 1.8 16.7 f 2.3
1.00 1.08 1.02 0.91
8.9 f 1.4 9.6 f 1.5 11.6 f 1.7 16.9 f 2.2
1.00 1.01 0.95 0.91
6.3 f 1.5 8.6 f 2.0 12.6 f 2.5 20.3 f 4.0
1.00 0.77 0.75 0.54
5.6 f 1.5 7.3 f 1.3 11.6 f 1.9 19.3 f 3.6
1.00
1.05 0.83 0.44
6.3 f 1.7 9.2 f 2.4 18.1 f 3.6 21.5 f 4.7
1.00 0.75 0.73 0.54
5.7 f 10.8 f 12.5 f 19.3 f
1.00 0.91 0.92 0.92
-
+
ARS8600-9007109 C 197 592 1775
9.1 f 11.0 f 12.9 f 17.6 f
1.3 1.6 2.1 3.2
1.00 0.83 0.59 0.37
C 137 412 1235
9.7 f 1.7 11.5 f 1.9 12.8 f 2.0 14.4 f 2.2
1.00 1.03 1.04 0.96
C 154 463 1398
6.3 k 1.4 5.5 f 1.3 5.2 f 1.3 7.3 f 1.8
1.00
-
+
-
+
+
+
-
+ +
ARS11500-900710 -
+
-
-
+ +
ARS28750-9004129 0.98 0.95 0.68
-
-
1.00
+ +
+
+
+ +
ARS51250-900822
f
1.5
1.00 0.96 0.62 0.52
332 997
8.9 f 10.2 k 11.5 f 12.2 f
1.3 1.4 1.5 1.6
1.00 0.96 1.04 0.95
C 128 384 1152
6.4 f 1.3 10.7 f 2.6 15.5 f 4.8 15.8 f 5.3
1.00 0.46 0.20 0.17
C 86 259 776
2.3 2.9 4.7 4.9
f
0.7
f 0.8 f 1.4
-
+
-
+
-
+ + +
-
+ +
CBKS7750-900822 C 111
-
-
+
-
-
+ +
+ +
EDDS2800-910225
+
-
NC NC
1.00
NC NC
+
h
EDDS5500-900412 C 51 154 461
5.9 f 1.3 5.5 f 1 . 2 5.8 f 1.4 7.1 f 1.5
1.00 1.16 1.02 0.92
C 69 208 625
4.4 f 5.2 f 4.8 f 5.0 f
1.1 1.1 1.0 1.0
1.00 1.23 1.33 1.35
C 72 217 652
7.4 f 1.1 12.5 f 3.1 13.2 f 3.2 13.6 f 2.8
1.00 0.23 0.23 0.33
C 139 417 1251
4.9 f 7.5 k 12.9 f 44.8 f
1.4 1.5 3.0
1.00
C 157 472 1415
6.0 f 7.6 f 8.7 k 11.5 f
1.3 1.6 1.9 2.4
0.76 0.66 0.56
C 35 106 319
9.7 f 1.7 13.6 f 2.6 30.0 f 5.7 30.3 k 6.7
1.00 0.86 0.37 0.27
I
-
-
+ +
-
+
FBKS0000-900806 -
-
+
+
HBKS0000-900412 NC NC
+
-
-
+
+
-
+
HBKS2500-8911109
11.2
0.89 0.43 0.16
-
+
-
NC
-
+ +
-
+
HBKS3500-9004128 1.00 -
+
-
-
+
+
+ + +
-
+
+
HPSS-900710
604
Environ. Sci. Technol., Vol. 26, No. 3, 1992
-
+
NC
+
1.4 2.1 2.2 2.9
-
+
Table I1 (Continued) -PMS"
concnC
mutant fractiond (Xl06)
relative survival'
C 187 561 1684
9.6 f 2.0 11.7 f 2.3 13.6 f 2.9 16.1 f 3.6
1.00 0.94 0.69 0.57
C 140 412 1264
10.2 f 1.6 7.0 f 1.5 10.5 f 2.3 10.8 f 2.6
1.00 0.71 0.54 0.42
C 103 310 930
4.4 f 1.6 3.6 f 0.8 4.4 f 1.0 4.0 f 1.0
1.00 1.35 1.17 1.03
C 121 363 1089
7.3 f 1.5 8.9 f 1.7 9.4 f 1.7 8.8 f 1.6
1.00 1.04 1.06 1.04
C 113 338 1014
7.0 f 1.2 6.5 f 1.2 9.1 f 2.4 7.7 f 1.3
1.00 0.90 0.49 0.88
C 57 170 511
6.3 f 1.4 7.7 f 1.4 7.3 f 1.4 9.0 f 1.6
1.00 1.13 1.09 1.15
+PMSb stat signif f HC
cc
mutant fractiond (X106)
relative survivale
cc
9.1 f 1.6 24.8 f 3.9 72.1 f 15.3 113.8 f 36.3
1.00 0.56 0.17 0.06
NC NC
9.5 f 1.9 11.9 f 2.5 17.0 f 3.4 20.4 f 4.0
1.00 0.77 0.75 0.54
8.9 f 1.4 13.1 f 1.8 15.0 f 2.1 18.7 f 2.6
stat signiff HC
LBKS5500-9008229 -
+
-
+
+
PHPSE-9008089 -
-
+
-
+
+
1.00 0.99 0.89 0.75
+ + +
-
12.5 f 1.6 17.4 f 2.0 23.2 f 2.7 24.8 f 2.7
1.00 0.99 0.83 0.87
+ + +
+ + +
5.3 f 1.5 9.8 f 1.9 16.4 f 2.9 20.4 f 3.0
1.00 1.16 1.09 1.19
+ + +
-
12.5 f 1.6 12.7 f 1.6 15.3 f 1.9 23.5 f 2.8
1.00 1.04 0.96 0.76
9.1 f 1.3 8.0 f 1.3 11.8 f 2.0 17.7 f 2.3
1.00 0.82 0.57 0.70
4.4 f 1.1 3.4 f 1.0 6.5 f 1.3 6.2 f 1.2
1.00 0.87 1.08 1.23
11.7 f 2.2 8.0 f 1.7 9.9 f 1.9 8.8 f 1.8
1.00 1.09 1.10 1.04
PHPSW-900808 -
-
-
+
UMLUFS-900710
-
WEPSE-900710
-
+
WEPSW-900710 -
-
+
+
WMPS-900710 C 29 87 262
9.1 f 1.3 10.8 f 2.5 13.8 f 4.6 13.2 f 3.7
1.00 0.33 0.13 0.19
C 2.5 7.5 22.4
11.0 f 1.8 11.0 f 1.6 11.5 f 1.6 12.7 f 1.6
1.00 1.28 1.28 1.56
C 14 43 128
14.9 f 2.0 12.1 f 1.9 13.4 f 2.1 14.7 f 2.3
1.00 0.86 0.79 0.79
NC NC
-
+
+
SOLVSB-900625' -
-
SOXSB-90080@ -
-
Extracts tested in the absence of postmitochondrial supernatant (PMS). *Extracts tested in the presence of postmitochondrial supernatant (PMS). Concentration is expressed as micrograms of sediment extract per milliliter of culture. C, concurrent negative control. Mutant cells per lo5 colony-forming cells f standard deviation. e Survival is measured relative to the negative control. f For an extract to be mutagenic, a mutant fraction must be greater than the concurrent negative control (CC) with 99% confidence and it must exceed the 95% upper confidence limit of the historical negative controls. For relative survival less than 0.33, mutagenicity is not interpreted, and result is designated NC or no call. g A precipitate formed in the DMSO during solvent exchange. "Despite high toxicity, number of induced colonies on mutation plates is greater than 2 X the number of colonies on control plates; therefore, test concentration is considered mutagenic. Solvent blank. jSystem blank.
WEPSW-910218 was not mutagenic, and at 100 pg/mL, relative survival was less than 50% (Table I, Figure 5). In comparing the results from the 72-h experiments with the results from the 28-h exposure experiments, it appears that extended exposure time did not result in a net increase in induced mutant fraction; however, the low survival suggests that extended exposure may have promoted the action of toxic chemicals present in the sediment extracts. B. S . typhimurium Assay. Of the 32 sediment samples tested for mutagenicity in the S. typhimurium forward mutation assay, 20 were found to be mutagenic
(Figure 2). All of the mutagenic samples tested positive in the presence of Aroclor-induced rat liver PMS. In addition, two samples, ARS8600 and HPSS, tested positive in the absence of PMS, indicating the presence of either direct-acting mutagens or chemicals such as nitropolycyclics, which are metabolized by bacterial enzymes. In five of the samples tested in the absence of PMS and in one sample tested in the presence of PMS, relative survival was too low for mutagenicity to be assessed. The results of mutagenicity and toxicity experiments for each of the mutagenic samples are shown in Table 11. System Environ. Sci. Technol., Vol. 26,
No. 3, 1992 605
J
02 !
2
0-
1 2-
0
A-.i.pi_pL-
20 40 60 80 Exposure Concentration (ug/mL)
100
Figure 5. Mutagenicity in MCL-3 cells induced by 72-h exposure to extracts of Wedge Pond sediments WEPSW-900821 and WEPSW910218. Each set of results is plotted as the mean mutant fraction f standard error for a single experiment performed in duplicate. For an exposure concentration to be mutagenic, the mutant fraction must be greater than the concurrent negative control (calculated using Dunnett's t test where p = 0.05),and it must exceed the 99 % upper confidence limit calculated from the historical negative control data base. No data points fulfilled both statistical criteria.
blanks were initially toxic in the assay in the presence of PMS; however, it was determined that residual amounts of the extraction solvents were present in the DMSO, and by improving solvent exchange efficiency, the toxicity was eliminated. The location of mutagenic sites in Figure 2 indicates that S. typhimurium mutagens are widely distributed in surface sediments in the watershed. Not only were samples which were collected near known hazardous waste sites (e.g., the East Drainage Ditch and Halls Brook) mutagenic under the conditions of the assay, but samples from apparently less contaminated areas [e.g., the headwaters of the Aberjona River (AR51250) and Fowle Brook (FBKOOOO)] were also mutagenic. This finding suggests that the principal S. typhimurium mutagens in the sediments are either ubiquitous anthropogenic chemicals (e.g., automobile exhaust emissions, pesticides, etc.) or naturally occurring sediment material such as humic substances. A third possibility is that both anthropogenic and naturally occurring S. typhimurium mutagens are present in the sediments. C. Metal Analysis. The concentrations of toxic metals in sediment samples ranged from below the limits of detection to maxima of 1000 mg/kg for As, 38 mg/kg for Cd, and 3400 mg/kg for Cr. P b ranged from 40 to lo00 mg/kg. As expected, high concentrations of metals were found in sediments near the Industriplex site. In three samples from South Pond, the average concentration of chromium (1360 mg/kg) was 3 times higher than the mean of all samples tested and the average concentration of lead (637 mg/kg) was twice the watershed average. Likewise, the average concentration of arsenic (420 mg/kg) in Halls Brook was 3 times higher than the watershed average. The sites where the highest levels of all four metals were detected (AR23400 and AR23600) are located l mile south 606
Environ. Sci. Technol.. Vol. 26, No. 3, 1992
of the Industriplex site, in a wetland area within the boundary of the Wells G&H site. Despite the large contrasts in metal concentrations from site to site, no significant correlations between mutagenicity and metal concentrations were observed. This may or may not mean that these metals played no role in inducing the observed mutagenicity. When neutral organic solvents were used in the sediment extraction procedure, it was expected that inorganic metal recoveries would be low (although organic metal species might be recovered more efficiently). However, to the extent that metal concentrations may be regarded as indicators of the level of industrial contamination, these results do not support the hypothesis that the source(s) of the mutagen(s) is(are) associated with the historical industrial activities responsible for toxic metal contamination in the watershed.
IV. Discussion When the results of the human lymphoblast and S. typhimurium assays are compared, the most compelling question is why the two sets of results are so different. In previous investigations in which pure compounds have been tested in both human lymphoblast and S. typhimurium assays, marked differences in sensitivities have been observed. Puju et al. (43) tested two N-nitroso bile acid conjugates, N-Nitrosotaurocholic acid and N-nitrosoglycoholic acid, in S. typhimurium (TM677) and human lymphoblast (TK6) mutation assays. It was observed that the two acids produced similar concentration-response curves with statistically significant mutant fractions at -0.12 mM in the bacteria assay; however, in the human cell assay, N-nitrosotaurocholic acid gave a statistically significant mutant fraction only at 0.04 mM, while Nnitrosoglycoholic acid was mutagenic at 0.05 pM, a dose 9000 times smaller. Penman et al. (44) compared the response of S. typhimurium (TM35) and human lymphoblast (MIT-2) assays to eight alkylating agents. It was found that bacterial cells were 3 times more sensitive to butyl methanesulfonate and 25 times more sensitive to P-propiolactone than human cells, but that human cells were 2.3-13 times more sensitive than S. typhimurium to methyl, ethyl, and propyl methanesulfonate, N-methylN'-nitro-N-nitrosoguanidine, methylnitrosourea, and Nmethyl-N-nitrosourethane. In addition, Deluca et al. (45) found that in testing the half mustard-substituted acridine ICR-191, the human lymphoblast cell line MIT-2 was 25 times more sensitive to ICR-191 than was S. typhimurium strain TA98. Deluca et al. hypothesized that the different responses in the two cell lines were linked to differences in the biochemical events required for mutation (e.g., cell reaction and response, DNA repair, etc.) and in the time of exposure to ICR-191 (28 h for the lymphoblasts vs 2 h for the bacteria). Considering these findings in which S. typhimurium and human lymphoblast assays have shown different sensitivities to pure compounds, it is therefore reasonable to expect that in testing complex mixtures the assays will show considerably different sensitivities. The contrasting outcomes we observed could also be in part the result of differences in the metabolizing enzyme systems in the two assays. In the S. typhimurium assay, an exogenous metabolizing enzyme system made from activated rat liver homogenates (PMS) is added to the culture, while the MCL-3 cells are capable of endogenous synthesis of three cytochromes (P450IA1, P450IA2, and P450IIA3) and microsomal epoxide hydrolase. Although the composition of Aroclor-induced enzymes present in rat liver PMS has not yet been fully characterized, it is known that PMS contains several different oxidative enzymes including the cytochromes P450IA1, P450IA2, P450IIA1,
P450IIB1, and P450IIB2 (46). Due to the differences in the composition and properties of enzymes in rat liver and human lymphoblasts, it is likely that different metabolites are produced as a result of exposing the two cell lines to promutagens present in sediment extracts. A third factor which could help explain the differences in the mutagenicity results from the two assays is solubility limitations of the sediment extracts in the cell cultures. In treating the human lymphoblast cultures with sediment extracts in DMSO, we observed that at doses in excess of 100 pg of extracted sediment solids/mL of cell culture, the cultures became turbid. This suggests that substances in the extracts were not soluble in the culture at the highest test concentrations. By contrast, in the S. typhimurium assay, precipitate formation was not observed in cultures, even at test concentrations in excess of 1mg/mL. These qualitative observations suggest that the actual range of dissolved sediment extract concentrations to which the human lymphoblasts were exposed was smaller than the concentration range to which S. typhimurium were exposed. A factor which could further confound the interpretation of our observations is the presence of mutation-inhibiting agents in the sediment extracts. It is likely that naturally occurring chemicals such as humic substances, are present in the extracts far in excess of pollutant concentrations. Conceivably, these compounds could selectively adsorb hydrophobic mutagens (thus making them biologically unavailable), limit the metabolic activation of promutagens by binding with oxidative enzymes, or themselves be activated and preferentially bind with nucleophilic sites on DNA. Recent results obtained by Mukherjee (47), for instance, indicate that crude extracts of lignite tars are less mutagenic in the human lymphoblast (MCL-3) assay than would be expected, based on the benzo[a]pyrene and cyclopenta[cd]pyrene content of the tar. Interestingly, this apparent inhibitory effect was not evident in testing the same extracts a t low doses in S. typhimurium (TM677); however, when higher concentrations from a second lignite tar sample were tested in S. typhimurium, mutation inhibition was again observed. Subsequent testing of extracts of wood pyrolysis tars produced a similar inhibitory effect. On the basis of these results, Mukherjee hypothesized that nonmutagenic lignite and wood tar constituents, such as quinones (a class of known human lymphoblast mutation inhibitors), were inhibiting the activity of mutagenic constituents.
V. Conclusions This work demonstrates that mutagens for both S. typhimurium and human lymphoblasts are present in sediments on the Aderjona watershed. When extracts of 32 sediment samples collected from sites throughout the watershed were tested, 20 samples produced statistically significant mutagenicity in a S. typhimurium forward mutation assay, and two samples produced statistically significant mutagenicity in a human lymphoblast mutation assay. The two samples which were mutagenic in human lymphoblasts were also mutagenic in S. typhimurium. In addition, six samples were highly toxic (Le., >67% killing) in S. typhimurium, and the highest doses from 12 samples were so toxic to human lymphoblasts that the treated cultures could not be assayed for induced mutation. Mutagenicity and toxicity induced by sediment extracts was not significantly correlated with metal content, and the distribution of sediments in the watershed which were mutagenic was not strongly associated with proximity to known industrial waste disposal sites. These findings were surprising. It was expected that sediments in depositional
areas downstream from the two CERCLA sites and other industrial areas in the watershed would serve as traps for anthropogenic mutagens. Instead, the results indicate that S. typhimurium mutagens are widely distributed, and that human lymphoblast mutagens are present a t sites that would not have been predicted a priori to be among the more highly contaminated sites in the watershed. Additional research is necessary to elucidate the chemical nature of mutagens present in Aberjona watershed sediments. An appropriate next step would be to fractionate mutagenic samples, thereby separating classes of mutagens into chemically distinct subsamples and, thus, facilitating chemical analysis. Although S. typhimurium mutagens are more widely distributed throughout the watershed, the finding that sediment samples from Wedge Pond and Little Brook were mutagenic in human lymphoblasts should be regarded as more direct evidence that these sediments could pose health risks to humans. Given the goal of identifying environmental chemicals suspected of causing detrimental human health effects, we believe that sediments from these two sites should be the focus of future efforts to isolate and identify specific mutagens. Acknowledgments We thank Jennifer Zemach and Kim Heroy for their assistance in preparing sediment samples, Henny Smith and Woody Bishop for performing the S. typhimurium assays, the researchers at Gentest Corp., who tested our samples in the human lymphoblast assay, and Henry Spliethoff for determining metals content. Literature Cited (1) Kool, H. J.; Van Kreijl, C. F. Sci. Total Enuiron. 1988, 77, 51-60. (2) Maruoka, S.; Yamanaka, S.; Yamamoto, Y. Sci. Total Enuiron. 1986, 57, 29-38. (3) Maruoka, S.; Yamanaka, S.; Yamamoto, Y. Water Res. 1985, 19, 249-256. (4) Slooff, W.; Van Kreijl, C. F. Aquat. Toxicol. 1982,2,89-98. ( 5 ) Van Hoof, F.; Verheyden, J. Sci. Total Environ. 1981,20, 15-22. (6) Pelon, W.; Whitman, B. F.; Beasley, T. W. Enuiron. Sci. Technol. 1977,11,619-623. (7) Osborne, L. L.; Davies, R. W.; Dixon, K. R.; Moore, R. L. Water Res. 1982, 16, 899-902. (8) Moore, R. L.; Osborne, L. L.; Davies, R. W. WaterRes. 1980, 14,917-920. (9) West, R. W.; Smith, P. A.; Booth, G. M.; Wise, S. A.; Lee, M. L. Arch. Enuiron. Contam. Toxicol. 1986,15,241-249. (10) West, R. W.; Smith, P. A.; Booth, G. M.; Lee, M. L. Environ. Toxicol. Chem. 1986,5, 511-519. (11) Sato, T.; Kato, K.; Ose, Y.; Nagase, H.; Ishikawa, T. Mutat. Res. 1985, 157, 135-143. (12) Sato, T.; Momma, T.; Ose, Y.; Ishikawa, T.; Kato, K. Mutat. Res. 1983,118, 257-267. (13) Fabacher, D. L.; Schmitt, C. J.; Besser, J. M.; Mac, J. M. Enuiron. Toxicol. Chem. 1988, 7, 529-543. (14) Samoiloff, M. R.; Bell, J.; Birkholz, D. A.; Webster, G. R. B.; Arnott, E. G.; Pulak, R.; Madrid, A. Enuiron. Sci. Technol. 1983,17, 329-334. (15) Grifoll, M.; Solanas, A. M.; Bayona, J. M. Arch. Enuiron. Contamin. Toxicol. 1990, 19, 175-184. (16) Suzuki, J.; Sadamasu, T.; Suzuki, S. Enuiron. Pollut., Ser. A 1982,29, 91-99. (17) Hirayama, K.; Suzuki, J.; Suzuki, S. Jpn. J. Limnol. 1981, 42, 82-88. (18) Kinae, N.; Hashizume, T.; Makita, T.; Tomita, I.; Kimura, I.; Kanamori, H. Water Res. 1981, 15, 17-24. (19) Oishi, S.; Takahashi, 0. Bull. Environ. Contam. Toxicol. 1987, 39, 696-700. (20) Pelon, W.; Beasley, T. W.; Lesley, D. E. Enuiron. Sci. Technol. 1980, 14, 723-726. Environ. Sci. Technol., Vol. 26, No. 3, 1992
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Received for review May 17, 1991. Revised manuscript received October 24,1991. Accepted October 29,1991. This research was supported by NIEHS Grant 1 -P42-ES04675 (Superfund Basic Research) and the MIT Environmental Health Sciences Center Grant (2-P30-ES02109-13).
