Wiley

The use of polymers in plastic and rubber products has generated concern that monomers potentially active in biological systems may be eluted from these substances. We have evaluated two such monomers, acrylonitrile and styrene, for the induction of chromosome damage in mice. Butadiene monoxide, a presumed metabolite of a third important monomer, 1,3‐butadiene, was also tested. These chemicals were administered as a single intraperitoneal injection; sister chromatid exchanges and chromosome aberrations were analyzed in bone marrow cells. Acrylonitrile and styrene were largely negative for these endpoints when tested at doses ranging to 60 mg/kg and 1,000 mg/kg, respectively. Butadiene monoxide, which previously has not been tested in a mammalian system, was determined to be a very effective inducer of sister chromatid exchanges and chromosome aberrations. Both endpoints showed a clear dose response and a greater than ten‐fold increase over control levels at high doses. These studies represent an initial step in our efforts to evaluate genetic risk associated with exposure to common polymeric chemicals.

The incidences of chromosome aberrations and the frequencies of sister chromatid exchanges (SCE) were investigated in cultured lymphocytes of 18 styrene‐exposed workers in comparison with six controls. There was a marginal increase in the incidence of structural chromosomal aberrations in first‐division metaphases in the styrene‐exposed workers, as compared with the nonexposed controls. However, there was no difference in SCE frequencies. When each group was divided into smokers and nonsmokers, styrene‐exposed smokers tended to have higher SCE frequencies than styrene‐exposed nonsmokers. Furthermore, cell proliferation was inhibited in styrene‐exposed workers (both smokers and nonsmokers) and control smokers.

Groups of male B6C3F1 mice (N = 12) were exposed to ambient air or to gaseous 1,3‐butadiene (BD) at 6.25, 62.5, and 625 ppm for 10 exposure days (6 hr + T₉₀/day). Exposure to BD induced in bone marrow: 1) a significant increase in the frequency of chromosomal aberrations (CA); 2) a significant elevation in the frequency of sister chromatid exchanges (SCE); 3) a significant lengthening of the average generation time (AGT); 4) a significant depression in the mitotic index (MI); and, as measured in the peripheral blood, 5) a significant increase in the proportion of circulating polychromatic erythrocytes (%PCE), and 6) a significant increase in the level of micronucleated PCE (MN‐PCE) and micronucleated normochromatic erythrocytes (MN‐NCE). The most sensitive indicator of genotoxic damage was the frequency of SCE (significant at 6.25 ppm), followed by MN‐PCE levels (significant at 62.5 ppm), and then by CA and MN‐NCE frequencies (significant at 625 ppm). The most sensitive measure of cytotoxic damage was AGT (significant at 62.5 ppm), followed by %PCE (significant at 625 ppm), and then by MI (significant by trend test only). Because each cytogenetic endpoint was evaluated in every animal, a correlation analysis was conducted to evaluate the degree of concordance among the various indicators of genotoxic and cytotoxic damage. The extent of concordance ranged from a very good correlation between the induction of MN‐PCE and the induction of SCE (correlation coefficient r = 0.9562) to the lack of a significant correlation between the depression in the MI and any other endpoint (r < 0.37).

A number of metal compounds have been shown to be human carcinogens. Others, while not proven human carcinogens, are able to cause tumors in laboratory animals. Short‐term bacterial assays for genotoxic effects have not been successful in predicting the carcinogenicity of metal compounds. We report here the ability of some metal compounds to cause the induction of λ prophage in E coli WP2_S(λ). By far the strongest inducing ability was observed with K2CrO4, followed by Pb(N0₃)₂ > MnCl₂ > Ni(OOCCH₃)₂ > CrCl₂ > NaWO₄ > Na₂MoO₄ > KMnO₄. With the exception of chromate, long‐term exposures in a narrow, subtoxic dose range were required in order to demonstrate phage induction. A new microtiter assay for λ prophage induction, which incorporates these features, is described. This system also was able to detect very small amounts of organic carcinogens.

The utility of the sister chromatid exchange (SCE) assay for human population studies is potentially limited by the variability associated with individual baseline SCE frequencies. This investigation identifies and quantifies the major sources of preparative and biological variation associated with the determination of baseline SCE frequencies in cultured human lymphocytes. Much of the variation in lymphocyte SCE frequencies is attributable to the amount of bromodeoxyuridine (BrdUrd) available per lymphocyte; the pooled coefficient of variation (CV) over the dose range of 10 to 160 μM is about 18%. Other variations in the baseline frequency result from culture‐to‐culture and slide‐to‐slide differences. The pooled coefficient of variation among donors is about 10%. The effect of cell‐to‐cell differences in baseline SCE frequency among donors can be minimized by increasing the number of cells scored per donor. When 20 cells are analyzed per individual the pooled cell‐to‐cell variation is 9% but when 40 or 80 cells are analyzed it is reduced to 6 and 4% respectively. For a single individual the cell‐to‐cell coefficient of variation at 100 μM BrdUrd is 40.8%. Under our experimental conditions, a 30% increase in SCE frequency between two cohort populations can be detected with a 95% probability at a 5% level of significance when 11 individuals per cohort are studied. For a longitudinal or in vitro dose response study of a single individual, a 50% increase in SCE frequency can be detected with a 95% probability at a 5% level of significance when 25 cells per sample are analyzed. These results indicate the feasibility of applying the SCE bioassay to humans as a measure of environmental stress.

Ethylene oxide is a known mutagen as indicated by short‐term testing in vitro and in vivo. Occupational exposure can occur during ethylene oxide gas sterilization of materials for hospital and other use. To study the problem in a hospital sterilization facility where occupational exposure was suspected, epidemiologic, analytic, and bioassay tools were employed. All persons whose work activities involved some aspect of the sterilization process were considered exposed to the gas. Within this group of symptomatic and asymptomatic individuals, chronic and incidental exposure was documented by clinical history. Sister chromatid exchanges were studied in lymphocytes cultured from exposed individuals as well as comparable controls. Four chronically exposed persons who reported upper respiratory and neurologic symptoms were studied in some detail. This group showed significantly increased sister chromatid exchange at three weeks and again at eight weeks after the last known exposure. Another group of eight persons with fewer complaints studied as late as the ninth week showed significantly increased exchanges. Incidental exposure may also increase sister chromatid exchange. The measured maximum concentration of ethylene oxide in the sterilizer room was 36 ppm (within standards set by the Occupational Safety and Health Administration).

Ethylene oxide, which is the simplest epoxide and an extremely important commercial compound, has been used by many investigators as a model compound to study mutagenicity by alkylation of DNA. Knowledge of in vivo dose‐effect relations under experimental conditions may provide further insight into the dynamics of the sister chromatid exchange (SCE) response. It may also provide information on temporal aspects of sampling design for human worker populations. Groups of four male New Zealand white rabbits were exposed in inhalation chambers to 0, 10, 50, and 250 parts per million (ppm) ethylene oxide for 6 hr a day, 5 days a week, for 12 weeks. Peripheral blood samples were taken before the start of exposure, at intervals during exposure, and up to 15 weeks after the end of exposure to measure SCE rates in peripheral lymphocytes as well as standard hematological endpoints. Additionally, the level of reduced glutathione (GSH) in liver and blood was measured in a set of concurrently exposed animals at the end of the 12‐week exposure. Results show that exposure to 10 ppm does not cause a detectable increase in SCE rates. However, exposure to 50 and 250 ppm does cause an increase in SCEs that decreases after exposure ends, but still remains above baseline levels 15 weeks after exposure. Hematological and GSH measurements did not differ between control and exposed groups. These results indicate that inhalation exposure to the mutagenic alkylating agent ethylene oxide results in a dose‐related SCE effect, and that SCE is a more sensitive indicator of exposure than either standard hematological end points or GSH levels.

The mutagenicity of several organophosphorus pesticides (OPP) has been demonstrated by Wild [1975] in microorganisms. However, the results as reported in the literature of cytogenic, mutagenic, and carcinogenic tests using mammalian cells or experimental animals were scanty and inconclusive [Huang, 1973; Wild, 1975]. This may be partly due to the high toxicity and rapid degradation of these compounds by mammalian cells both in vitro and in vivo [Wild, 1975]. Since sister chromatid exchange (SCE) has proved to be a highly sensitive indicator of genetic damage [Latt, 1975] and for certain chemicals there was a good correlation between the induction of SCE and point mutations in mammalian cells [Carrano et al, 1978; Sirianni and Huang, 1980], we have studied and reported the effect of a total of 17 OPP on induction of sister chromatid exchange and cell cycle delay in an in vitro system using V79 cells without metabolic activation [Chen et al, 1981, 1982]. Among the 17 OPP tested, 8 were able to induce increased SCE frequencies and the other 9 caused no increase in SCE as compared to the control values. The OPP that proved to be positive SCE inducers were methyl‐parathion, demeton, trichlorfon, dimethoate, malathion, methidathion, oxydemeton methyl, and fenthion. The other nine that caused no SCE increase were diazinion, disyston, amaze, azinphosmethyl, bolstar, DEF, fensulfothion, monitor, and nemacur. In addition, cell cycle delay in various degrees was observed after treatment of V79 cells with all the OPP except for fensulfothion and oxydemeton methyl. However, for all experiments there was no clear correlation between the degree of cell cycle delay and induction of SCE.

A novel test system for the detection of mutagenic and recombinogenic activity of chemicals is described in detail. Drosophila melanogaster larvae trans‐heterozygous for the mutations multiple wing hairs (mwh) and flare (fir) are exposed to the test compounds for various periods of time ranging from 96 hr to 1 hr. Induced mutations are detected as single mosaic spots on the wing blade of surviving adults that show either the multiple wing hairs or flare phenotype. Induced recombination leads to mwh and fir twin spots and also to a certain extent, to mwh single spots. Recording of the frequency and the size of the different spots allows for a quantitative determination of the mutagenic and recombinogenic effects. This and earlier studies with a small set of well‐known mutagens indicate that the test detects monofunctional and polyfunctional alkylating agents (ethyl methanesulfonate, diepoxybutane, mitomycin C, Trenimon), mutagens forming large adducts (aflatoxin B₁), DNA breaking agents (bleomycin), intercalating agents (5‐aminoacridine, ICR‐170), spindle poisons (vinblastine), and antimetabolites (methotrexate). In addition, the test detects mutagens unstable in aqueous solution β‐propiolactone), gaseous mutagens (1,2‐dibromoethane), as well as promutagens needing various pathways of metabolic activation (aflatoxin B₁, diethylnitrosamine, dimethylnitrosamine, mitomycin C, and procarbazine). The rapidity and ease of performance as well as the low costs of the test necessitate a high priority for validation of this promising Drosophila short‐term test.