Showing papers in "Environmental Mutagenesis in 1986"

Salmonella mutagenicity tests: II. Results from the testing of 270 chemicals.

Abstract: This publication includes data of Salmonella mutagenicity results on 270 coded chemicals, encompassing 329 tests performed by three laboratories under contract to the National Toxicology Program (NTP). The preincubation modification of the Salmonella/mammalian microsome assay was used to test chemicals in up to five Salmonella strains in the presence and absence of rat and hamster liver S-9. With a few exceptions, inter- and intralaboratory reproducibility was good.

Chemically induced aneuploidy in mammalian cells: Mechanisms and biological significance in cancer

Abstract: A growing body of evidence from human and animal cancer cytogenetics indicates that aneuploidy is an important chromosome change in carcinogenesis. Aneuploidy may be associated with a primary event of carcinogenesis in some cancers and a later change in other tumors. Evidence from in vitro cell transformation studies supports the idea that aneuploidy has a direct effect on the conversion of a normal cell to a preneoplastic or malignant cell. Induction of an aneuploid state in a preneoplastic or neoplastic cell could have any of the following four biological effects: a change in gene dosage, a change in gene balance, expression of a recessive mutation, or a change in genetic instability (which could secondarily lead to neoplasia). To understand the role of aneuploidy in carcinogenesis, cellular and molecular studies coupled with the cytogenetic studies will be required. There are a number of possible mechanisms by which chemicals might induce aneuploidy, including effects on microtubules, damage to essential elements for chromosome function (ie, centromeres, origins of replication, and telomeres), reduction in chromosome condensation or pairing, induction of chromosome interchanges, unresolved recombination structures, increased chromosome stickiness, damage to centrioles, impairment of chromosome alignment, ionic alterations during mitosis, damage to the nuclear membrane, and a physical disruption of chromosome segregation. Therefore, a number of different targets exist for chemically induced aneuploidy. Because the ability of certain chemicals to induce aneuploidy differs between mammalian cells and lower eukaryotic cells, it is important to study the mechanisms of aneuploidy induction in mammalian cells and to use mammalian cells in assays for potential aneuploidogens (chemicals that induce aneuploidy). Despite the wide use of mammalian cells for studying chemically induced mutagenesis and chromosome breakage, aneuploidy studies with mammalian cells are limited. The lack of a genetic assay with mammalian cells for aneuploidy is a serious limitation in these studies.

Statistical analyses for in vitro cytogenetic assays using Chinese hamster ovary cells

Abstract: It is a widely held view that objective statistical criteria are needed for the evaluation of genetic toxicity assays. This paper presents statistical methods for the analysis of data from in vitro sister chromatid exchange (SCE) and chromosome aberration tests that use Chinese hamster ovary cells. For SCEs, an extensive study of solvent control results demonstrated that there is a substantial interday component of variability in the data, and that a Poisson sampling model is applicable to data generated via the protocol of Galloway et al [1985]. Consequently, a trend test for evidence of a dose response is proposed for such SCE data. As an illustration of this statistical method, analysis of data previously considered to be negative [Gulati et al, 1985] indicates that di(2-ethyl-hexyl) phthalate induces a weak, but reproducible, SCE dose response in CHO cells. Monte Carlo methods are used to show that the trend test is more sensitive than four other statistical procedures considered for the analysis of Poisson-distributed SCEs. A similar trend test for dose response in proportions is proposed for chromosome aberration data, where the percent of cells with chromosome aberrations is the response of interest. Sensitivity (or power) studies indicate that three doses and a control with 50 cells/dose point is a reasonable design for an in vitro SCE study that uses the Galloway et al protocol. For in vitro chromosome aberrations, however, three doses and a control with 100 cells/dose point appears to produce too insensitive an assay; an increase to 200 cells/dose point in the Galloway et al protocol seems worthy of serious consideration.

Mutagenicity of fine (< 2.5 μm) airborne particles: Diurnal variation in community air determined by a salmonella micro preincubation (microsuspension) procedure

Abstract: A simple modification of the Salmonella liquid incubation assay previously developed for detecting mutagens in urine was used to determine mutagenic activity of airborne particulate matter. The modification consists of adding ten times more bacteria (approximately 10(9) per incubation tube) and five to ten times less metabolic enzymes compared to the plate incorporation method. The mixture volume is approximately 0.2 ml, and the mixture is incubated for 90 min before pouring it according to the standard protocol. The modified procedure (micro preincubation or microsuspension) was approximately ten times more sensitive than the standard plate incorporation test for detecting mutagens in air particulate extracts and approximately ten to 31 times more sensitive for the chemical mutagens 2-nitrofluorene, 4-nitroquinoline-N-oxide, 2-aminofluorene, and benzo(a)pyrene in bacterial strain TA98. Mutagenic activity was detected in particle extracts obtained from 1 m3 of air (17 micrograms of extract) or less. This microsuspension procedure was applied to air particulate samples collected with low-volume (15-50 liters per min) virtual-dichotomous air samplers. Mutagenic activity was associated exclusively with fine particles (aerodynamic diameters of less than 2.5 microns). Diurnal patterns of mutagenic activity (TA98 revertants per cubic meter air) were investigated by measuring filter extracts from 2-hr samples collected in threemore » San Francisco Bay Area cities during the summer or fall of 1982. Four criteria pollutants--lead, nitrogen dioxide, ozone, and sulfur dioxide--were simultaneously sampled at one location. Mutagenicity from fine particles sampled at this location was highly correlated with lead and much less correlated with nitrogen dioxide, ozone, and sulfur dioxide. The microsuspension procedure is applicable in testing samples of limited mass.« less

Genotoxic effects in cultured mammalian cells produced by low pH treatment conditions and increased ion concentrations.

Abstract: A workshop describing the mutagenic effects of low pH and high osmotic levels on cultured mammalian cells was held at the 16th Annual Meeting of the Environmental Mutagen Society in Las Vegas, Nevada, 1985. Data presented in this summary report were generously provided by the five invited speakers.

Classifying mutagens as to their specificity in causing the six possible transitions and transversions: a simple analysis using the Salmonella mutagenicity assay.

Abstract: The standard Salmonella tester strains used to detect base substitution mutations carry the hisG428 ochre mutation (TA102 and TA104) and the hisG46 missense mutation (TA100). These mutations can be reverted by base changes at their mutant his loci or at extragenic suppressor loci. The base changes resulting in each class of revertants of these mutations have been identified, and simple phenotypic screens have been developed to distinguish among them. Revertants at extragenic suppressor loci are distinguished from those at the his loci by their sensitivity to inhibitory histidine analogs. The four ochre suppressor loci of hisG428 are distinguished by their ability to support growth of nonsense mutants of phage P22. These screens are the basis for a rapid and simple system for determining the base substitution specificity of mutagens using hisG428- and hisG46-containing tester strains. Diagnostic mutagens specific for each of the six possible base changes (transitions and transversions) have been identified. Using these diagnostic mutagens, two additional strains, each specifically reverted by a single base substitution mutation, have been developed to provide a minimum of two loci at which to detect each type of base change. The ability of this system to provide detailed information about mutational specificity in a variety of DNA repair backgrounds will allow further elucidation of the mechanisms of mutagenesis and DNA repair.

Target sequences for mutagenesis in Salmonella histidine-requiring mutants

Abstract: Nucleotide target sequences involved in reversion to the wild type phenotype are diagrammed for Salmonella frameshift histidine-requiring mutants hisD3052, hisD3018, hisD6610, and hisD6580 and for base-substitution mutants hisG46 and hisG428. Frameshift strain hisC3076 probably reverts by nucleotide changes similar to those that occur during reversion of hisD3018 and hisD6610. Multiple modes of reversion characterize each strain. Each strain also has a particularly diagnostic mutagen-susceptible sequence. These highly mutagen-susceptible stretches are the hisD3052 GCGCGCGC sequence, the hisD6610 CCCCCC sequence, the hisD6580 AAAAA sequence, and the A/T containing codon of hisG428 and G/C containing codon of hisG46, respectively. Between them, hisG46 and hisG428 are reverted by all of the six possible base substitution transition and transversion mutations.

Structure-activity relationships (SARs) among mutagens and carcinogens: a review.

Abstract: This review is an introduction to methods for evaluating structure-activity relationships (SARs), and, in particular, to those methods that have been applied to study mutagenicity and carcinogenicity. A brief history and some background material on the earliest attempts to correlate molecular structure and biological activity are included. Most of the discussion focuses on modern methods utilizing extrather-modynamic and physical property variables such as the Hansch method and SIMCA, and approaches based on molecular connectivity such as the ADAPT, CASE, and Enslein methods. In general, the latter class is potentially the most useful in the study of the large and structurally diverse databases so often encountered in the study of mutagenicity and carcinogenicity. They also are not very sensitive to lab-to-lab variances in reported activities and outright misclassifications in activities of some compounds. This is chiefly because the statistical treatments used in these methods tend to dilute the importance of outliers. The methods using physicochemical and extrathermodynamic variables are especially important in relatively small, congeneric databases and can help fine-tune the role of physicochemical properties in mechanistic hypotheses. All of the above methods have been used to look at mutagenicity and carcinogenicity and some of the results reported in the literature are reviewed here. As far as specific methods go, ADAPT, CASE, SIMCA and the Enslein approach all seem to have similar classification powers (in the range of 75–95%), depending very much on the database studied. The emphasis in this review is on showing that the use of these computer-aided storage, retrieval and analysis techniques is a timely approach to predicting and even understanding the toxicity of environmental substances. However, each of the methods discussed is still under development, and their potential usefulness for predictive purposes is still being explored.

Sodium arsenite potentiates the clastogenicity and mutagenicity of DNA crosslinking agents

Abstract: To see if sodium arsenite enhances the clastogenicity and the mutagenicity of DNA crosslinking agents, Chinese hamster ovary (CHO) cells and human skin fibroblasts were exposed to cis-diamminedichloroplatinum (II) (cis-Pt(II)) or 8-methoxypsoralen (8-MOP) plus long-wave ultraviolet light (UVA) and then to sodium arsenite. The results indicate that the clastogenicity of cis-Pt(II) and 8-MOP plus UVA are enhanced by the post-treatment with sodium arsenite. Chromatid breaks and exchanges are predominantly increased in doubly treated cells. Furthermore, the mutagenicity of cis-Pt(II) at the hypoxanthine-guanine phosphoribosyl transferase locus is also potentiated by sodium arsenite in CHO cells.

Chromosome analysis of trifluorothymidine‐resistant L5178y mouse lymphoma cell colonies

Abstract: Cells from small (sigma) and large (lambda) trifluorothymidine-resistant (TFTr) colonies induced by chemical mutagen treatment of TK+/-L5178Y mouse lymphoma cells were examined for chromosomal abnormalities. Analysis of G-banded metaphase chromosomes from 34 sigma-TFTr colonies revealed that cells from 20 (59%) possessed one or more chromosomal abnormalities. The most frequent (16/20 colonies) abnormality observed in cells from sigma-TFTr colonies involved the addition of extra chromatin to the distal region of one chromosome number 11. In 13 of these 16 colonies, the origin of the chromatin translocated to chromosome number 11 could not be identified; the chromatin was not missing elsewhere in the genome. The remaining three sigma-TFTr colonies with an abnormal chromosome number 11 had apparently whole chromosomes translocated, in tandem, to the distal region of chromosome number 11. Chromosomal abnormalities observed in cells from sigma-TFTr colonies with normal number 11 chromosomes included 2N/4N and 2N/4N/8N mosaicism (two colonies), a Robertsonian translocation involving chromosome 10 and a marker chromosome (one colony), and trisomy 7 (one colony). In most (14/16) sigma-TFTr colonies with structural damage to chromosome number 11, the cells within a colony were heterogeneous in that some possessed chromosomal damage whereas others were apparently normal. Analysis of chromosomes in cells from eight lambda-TFTr colonies revealed one colony in which all cells had a Robertsonian translocation involving chromosomes 1 and 16 plus other structural abnormalities. The chromosomes of cells from the remaining lambda-TFTr colonies were apparently normal.

Sister chromatid exchange and chromosome aberration analyses in mice after in vivo exposure to acrylonitrile, styrene, or butadiene monoxide.

Abstract: 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.

Ethylene oxide dose and dose‐rate effects in the mouse dominant‐lethal test

Abstract: In the dose-response study, male mice were exposed by inhalation to ethylene oxide (EtO) for 4 consecutive days. Mice were exposed for 6 hr per day to 300 ppm, 400 ppm, or 500 ppm EtO for a daily total of 1,800, 2,400, or 3,000 ppm X hr (total exposures of 7,200, 9,600 and 12,000 ppm X hr), respectively. In the dose-rate study, mice were given a total exposure of 1,800 ppm X hr per day, also for 4 consecutive days, delivered either at 300 ppm in 6 hr, 600 ppm in 3 hr, or 1,200 ppm in 1.5 hr. Quantitation of dominant-lethal responses was made on matings involving sperm exposed as late spermatids and early spermatozoa, the most sensitive stages to EtO. In the dose-response study, a dose-related increase in dominant-lethal mutations was observed, the dose-response curve proved to be nonlinear. In the dose-rate study, increasing the exposure concentrations resulted in increased dominant-lethal responses.

Induction of SOS genes of Escherichia coli by chromium compounds.

Abstract: The induction of several SOS genes of Escherichia coli such as recA, umuC, and sfiA by hexavalent (K/sub 2/Cr/sub 2/O/sub 7/, K/sub 2/CrO/sub 4/, and CrO/sub 3/) and trivalent (CrCl/sub 3/, Cr(NO/sub 3/)/sub 3/, and (CH/sub 3/COO)/sub 3/Cr) compounds of chromium was studied. Induction was measured as ..beta..-galactosidase activity, using lacZ gene fusions under the control region of different SOS genes. The hexavalent chromium forms induced the genes responsible for massive synthesis of RecA protein, error-prone repair, and inhibition of cell division. On the other hand, the trivalent chromium compounds were unable to induce any of the SOS genes tested. Individual assay of hexavalent chromium compounds showed that K/sub 2/Cr/sub 2/O/sub 7/ was a stronger inducing agent of those three SOS genes tested than K/sub 2/CrO/sub 4/, which, in turn, was stronger than CrO/sub 3/. All this data led to the conclusion that hexavalent chromium compounds, but not trivalent, are proficient agents of induction of the SOS system and can produce indirect mutagenesis in Escherichia coli.

Induction of cytogenetic damage in rodents after short‐term inhalation of benzene

Abstract: Experiments were designed to investigate both the induction of sister chromatid exchanges (SCEs) in peripheral blood lymphocytes (PBLs) and micronuclei (MN) in bone marrow polychromatic erythrocytes (PCEs) of mice and rats after inhalation of benzene (BZ). Male DBA/2 mice (17-19 weeks old) were exposed to target concentrations of either 0, 10, 100, or 1,000 ppm BZ for 6 hr. Male Sprague-Dawley rats (11-14 weeks old) were exposed to target concentrations of either 0, 0.1, 0.3, 1, 3, 10, or 30 ppm BZ for 6 hr. Blood was obtained by cardiac puncture 18 hr after exposure, and PBLs were cultured in the presence of lipopolysaccharide (mouse B cells, 60 micrograms/ml) or concanavalin A (rat T cells, 30 micrograms/ml) to stimulate blastogenesis for SCE analysis. Femoral bone marrow smears from both species were analyzed for MN in PCEs 18 hr after BZ exposure. Mouse PBLs revealed a significant concentration-related increase in the SCE frequency over controls at 10, 100, or 1,000 ppm BZ. Mouse bone marrow showed a significant concentration-dependent increase in MN over controls after exposure to 10, 100, or 1,000 ppm BZ. Rat PBLs showed a significant increase in the SCE frequency after exposure to 3, 10, or 30 ppm BZ. The statistical significance of the 1 ppm BZ result was borderline and dependent on the statistical test chosen. Rat cells revealed a significant concentration-related increase in MN after inhalation of either 1, 3, 10, or 30 ppm BZ. PBLs from treated mice showed significant concentration-dependent decreases in mitotic indices; however, cell cycle kinetics and leucocyte counts remained unaffected. Rat PBLs showed significant decreases in mitotic activity only after exposure to 3 and 30 ppm BZ, whereas cell cycle kinetics and leucocyte counts were unaffected. These results show that BZ can induce statistically significant cytogenetic effects in PBLs and PCEs of both mice and rats after a 6-hr inhalation of BZ at low concentrations.

Comparison of multiple parameters of rodent carcinogenicity and in vitro genetic toxicity.

Abstract: The relationship between chemically induced patterns of tumorigenesis in rodents and of in vitro genetic toxicity was evaluated for 73 substances. tumorigenicity patterns were defined according to sex and species effects, the induction of common or uncommon tumors, and benign or malignant tumors. These results and the genetic toxicity results derived from the testing of chemicals under code were compared. Chemicals that induced tumors in both sexes of both rodent species (trans-sex/species carcinogens) were divided into those that showed multiple responses in genetic toxicity assays and those that showed little or no response. Some of the nongenotoxic trans-sex/species carcinogens exhibit properties that do not necessarily fit classification as only tumor promoters and may involve some other mode(s) of action. Those chemicals showing tumorigenicity in only one of the four groups exposed (uni-sex/species carcinogens) generally showed little or no response in genetic toxicity assays. Uni-sex/species carcinogens may be difficult to identify by in vitro assays because of their high tissue specificity. Chemicals that are tumorigenic in both sexes of both species are logically more likely to be tumorigenic in a third species than are those that are tumorigenic in only one sex of the exposed species. Therefore, while positive genetic toxicity test results are not predictive of all carcinogens, a consistent positive response among the multiple endpoints in these assays is more likely to identify chemicals with the potential for trans-sex/species carcinogenesis. Such trans-sex/species carcinogens may have the most direct implication for human health effects.

An interlaboratory comparison of transformation in Syrian hamster embryo cells with model and coded chemicals.

Abstract: Three independent laboratories tested eight “model” and five coded chemicals in the Syrian hamster embryo clonal transformation assay system to establish the intra- and interlaboratory reproducibility of the system and to identify sources of variability. When a common cell pool and the same lot of fetal calf serum were used, the three laboratories obtained consensus on the activity of eight model chemicals: five chemicals (benzo(a)pyrene, 7,12-dimethylbenz(a)anthracene, N-methyl-N′-nitro-N-nitrosoguanidine, nitroquinoline-N-oxide, and lead chromate) induced morphological transformation without exogenous metabolic activation and three (N-2-fluorenylacetamide, pyrene, and anthracene) produced no transformation response. Five coded chemicals (2,6-dichloro p-phenylenediamine, 4,4′-oxydianiline, cinnamyl anthranilate, dichlorvos, and reserpine), representative of environmental chemical classes, but not necessarily strong carcinogens, produced more equivocal responses in this interlaboratory study. Thus, while the assay can be used to distinguish between transforming and nontransforming chemicals in some cases, the intrinsic limitations in low transformation frequency and in achieving any dose-response results are major constraints to the use of this systsem in a routine testing program at the present time. Efforts to increase the transformation frequency or to amplify the expression of the transformed phenotype constitute some of the approaches which should be explored in order to overcome these limitations.

Glutathione: a protective agent in Salmonella typhimurium and Escherichia coli as measured by mutagenicity and by growth delay assays.

Abstract: Cultures of some aerobically grown strains of Salmonella typhimurium and Escherichia coli contain up to 24 microM extracellular glutathione (GSH) [Owens RO, Hartman PE (1985): Environ Mutagen 7(Suppl 3): 47] in addition to having intracellular GSH concentrations in the millimolar range. The addition of 26 microM GSH to cultures of Salmonella typhimurium strain TA1534 partially protected the bacteria from the toxic effects causing growth delay by 54 microM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). When MNNG was preincubated with equimolar GSH, the mutagenicity of the MNNG was neutralized. The addition of micromolar GSH to cultures of an Escherichia coli GSH- strain protected the cells from growth inhibition by micromolar concentrations of mercuric chloride, methyl mercuric chloride, silver nitrate, cisplatin, cadmium chloride, cadmium sulfate, and iodoacetamide. In the cases of mercuric chloride, cisplatin, MNNG, silver nitrate, and iodoacetamide, reaction products with GSH were detected by paper chromatography. In contrast to reduced GSH, micromolar concentrations of oxidized glutathione (GSSG) provided little or no protection and formed no detectable reaction products. Export of GSH by enteric bacteria may provide an important defense mechanism against exogenous toxic agents otherwise active in the micromolar range.

Measurement of in vivo mutant frequency in lymphocytes in the mouse

Abstract: A limiting-dilution cloning technique for quantifying in vivo mutations at the hypoxanthine phosphoribosyl transferase locus in mouse splenocytes was developed. Mouse splenocytes were cultured in round-bottom microwells with irradiated feeder cells, concanavalin A, and a source of interleukin 2 at five cells/well in the absence of thioguanine, and at 5 X 10(4) cells/well in the presence of 2.5 micrograms/ml thioguanine; mutant frequency was calculated as the ratio of the cloning efficiencies with or without thioguanine. The geometric mean (95% range) for the mutant frequency in 20 mice was 1.54 X 10(-6) (4.7 X 10(-7) -2.6 X 10(6)) and whole-body X-irradiation resulted in a dose-related increase in mutant frequency of up to approximately 20 times the baseline level. The in vivo murine mutation assay should be a useful system for genotoxicity testing and may be of particular value in establishing risk estimates for human populations exposed to genotoxins.

Micronuclei in red blood cells of the newt pleurodeles waltl after treatment with benzo(a)pyrene: Dependence on dose, length of exposure, posttreatment time, and uptake of the drug

Abstract: Aquatic larvae of the newt Pleurodeles waltl were exposed to different concentrations of benzo(a)pyrene (BaP) for various lengths of time. Frequencies of micronuclei in circulating erythrocytes were determined at different times after termination of the treatment. The incidence of micronuclei in larvae kept for 8 days in BaP-containing water displayed a marked increase with dose up to 0.075 ppm and a more gradual one with higher doses, reaching 158 per 1,000 at 0.75 ppm. The lowest dose at which a significant increase could be discerned was 0.01 ppm. Short periods of exposure, less than 2 days, did not result in a marked increase in micronuclei. Uptake and release was studied with tritiated BaP. Larvae concentrated BaP rapidly, attaining maximal levels after 12 hr. The ratio of radioactivity in larvae to that in an equivalent volume of surrounding water was about 200 independent of the amount of BaP added. Calf serum or bovine serum albumin added to the water lowered this ratio by competing for binding to BaP. Radioactive larvae placed in regularly renewed noncontaminated water lost 99% of the label after 100 hr. It is concluded that pleurodele larvae are a promising model for the detection of genotoxic activity in the aquatic environment.

Short-term test results for NTP noncarcinogens: an alternate, more predictive battery.

Abstract: A battery of short-term tests used to predict whether or not a chemical is a carcinogen must be both sensitive (correctly identifying carcinogens) and specific (correctly identifying noncarcinogens). A recent publication [Shelby and Stasiewicz, 1984, Environ Mutagen 6:871-876] of results in four short-term tests for 70 noncarcinogens tested under the aegis of the National Toxicology Program (NTP) indicates that the battery of short-term tests lacked specificity. We have analyzed these results using the Carcinogen Prediction and Battery Selection (CPBS) procedure and calculated that the specificity of the NTP battery is indeed very low, i.e., 0.50. Using published data from NTP, the Gene Tox program of EPA, and the collaborative study of the WHO International Programme on Chemical Safety, we have constructed an alternate battery that has fewer false positives; this battery has a specificity of 0.80. Thus, the lack of specificity of the original NTP battery does not imply that no set of short-term tests is able to predict carcinogenicity accurately.

Chromosome aberrations in mouse bone marrow cells following treatment in vivo with vinblastine and colcemid

Abstract: The present study was undertaken to determine if chemicals that are capable of inducing mitotic arrest and aneuploidy can also induce chromosomal breakage. Two chemicals, vinblastine and Colcemid, were selected to be studied. Their potentially clastogenic effects were investigated in mouse bone marrow cells in vivo. Two doses of vinblastine, 10(-5) M (9 mg/kg) and 10(-6) M (0.9 mg/kg), and one dose of Colcemid 10(-4) M (37 mg/kg), were administered to mice as single intraperitoneal injections. Bone marrow preparations were made at multiple time periods after injections, ie, 17, 24, 48, 72, and 96 hr. Both these agents, at the concentrations tested, induced mitotic arrest of bone marrow cells within 5 hr after injection. In recovering bone marrow cell populations, 2-3 days after drug administration, significantly elevated levels of chromosomal breakage (mainly the chromatid type) were observed. Vinblastine was found to be much more potent than Colcemid. Since both of these agents affect mainly microtubules, their actions to cause chromosomal breakage are likely to be indirect. Several possible clastogenic mechanisms such as interference with DNA synthesis, active metabolites, and cytoplasmic endonucleases are discussed.

Environmental tobacco smoke: comparative characterization by mutagenicity assays of sidestream and mainstream cigarette smoke.

Abstract: Mainstream cigarette smoke particles were collected by means of a smoking machine, and sidestream particles were collected from the room in which the smoking took place. The particles were extracted by sonication with acetone, and the extracts were solvent-exchanged to dimethyl sulfoxide. The samples were tested for mutagenicity in the Ames Salmonella/microsome assay. The mainstream extract is preferentially mutagenic in the presence of S9, with about 30,000 revertants/cigarette in TA98, but has little or no activity in its absence. The sidestream extract is also mutagenic in the presence of S9 with TA98, and this activity is mainly due to basic compounds. Sidestream smoke is also significantly mutagenic in the absence of S9 in the strain TA100 as well as in TA97 and TA104. This "direct" activity is due to components that are labile. The response of sidestream particles is 10,000-20,000 revertants/cigarette in TA98 + S9 and TA100-S9 when the collection is performed in a room where the particle concentration is modulated by deposition to surfaces. Sidestream particles collected on glass fiber filter and by electrostatic precipitation (ESP) with a commercial air cleaning device gave essentially the same mutagenic response, showing that ESP sampling may be an alternative to filter sampling for environmental tobacco smoke (ETS) in indoor environments. ESP sampling in children's rooms in smoking and nonsmoking homes showed that 5-10% of the tobacco smoke emitted in the smoking homes entered the child's room, demonstrating that diffusion of pollutants is faster than ventilation in modern buildings with low ventilation rates.

Dominant visible and electrophoretically expressed mutations induced in male mice exposed to ethylene oxide by inhalation.

Abstract: The offspring of DBA/2J male mice exposed to ethylene oxide (EtO) by inhalation had an increased incidence of both dominant visible and electrophoretically detected mutations over that found in control populations. The progeny at risk were obtained from matings during the exposure period and were the products of germ cells that were exposed throughout the entire spermatogenic process. The results reported here suggest that male germ cells repeatedly exposed to EtO during spermatogenesis are susceptible to EtO-induced transmissible damage.

Bacterial mutagenicity and chemical analysis of polycyclic aromatic hydrocarbons and some nitro derivatives in environmental samples collected in West Germany

Abstract: Snow and air particulate samples collected in Upper Frankonia, Federal Republic of Germany, have been analyzed for nitro-polycyclic aromatic hydrocarbons (PAH) and PAH content. A novel clean-up technique has been developed enabling interfering organochlorine environmental contaminants to be removed prior to analysis of the hydrocarbons by GC-MS. Mass fragmentation patterns are presented for 1-nitropyrene, 6-nitrobenzo(a)pyrene, 6-nitrochrysene, and 3-nitrofluoranthene. The level of these compounds found in air samples was in the range of 0.2–2.0 ng·m−3 with the exception of 6-nitrobenzo(a)pyrene, which was not detected. This compares with PAH values of between 1 and 6 ng.m−3. The freshly fallen snow sample collected at the side of a motorway had no detectable PAHs or nitro-PAHs. Parallel studies on the bacterial mutagenicity of the collected air samples using Salmonella typhimurium TA98 and TA100 in the presence and absence of aroclor-induced rat liver “S9” revealed both “direct” and “indirect” activity. Larger numbers of mutants were induced in the presence of S9 than in its absence. The snow sample was devoid of mutagenic activity. These studies show the utility of the biological approach to screen environmental samples prior to expensive and time-consuming chemical analysis.

Sister chromatid exchange induction and persistence in peripheral blood and spleen lymphocytes of mice treated with ethylnitrosourea

Abstract: The induction and persistence of sister chromatid exchanges (SCEs) were studied in peripheral blood and spleen lymphocytes of mice given a single i.p. injection of ethylnitrosourea (ENU) of 100, 350, or 600 muMoles ENU/kg. SCE frequencies were measured on days 1, 3, 5, and 7, and at seven additional times up to 172 days post-injection. SCEs were analyzed statistically by comparing the mean frequencies as well as the distribution of SCEs per cell at each time. The latter approach was based on a non-parametric method of identifying high frequency cells (HFCs). The SCE frequencies and proportion of HFCs in each dose and tissue remained elevated for up to 172 days following treatment, although the degree and statistical significance of the increase varied according to the tissue, dose, and statistical test employed. The SCE frequencies were found to oscillate during the first week. Following this, however, the return of the SCE frequencies to control levels was fit to a linear regression model with time as the only independent variable. The persistence of SCE-forming lesions was found to be dose-dependent for the spleen but not for blood. Within each dose the persistence of SCE-forming lesions was significantly greater for the blood relative to the spleen. The results emphasize that tissue, dose, and time since exposure are important factors to consider when quantifying SCEs in vivo; analysis of high frequency cells may be a more sensitive method of detecting exposure than the t-test; and a single determination of SCE frequencies may not be sufficient to quantitatively assess genotoxic damage in the first week following exposure.

The genetic toxicology of metal compounds: II. Enhancement of ultraviolet light-induced mutagenesis in Escherichia coli WP2.

Abstract: Salts of metals which are carcinogenic, noncarcinogenic, or of unknown carcinogenicity were assayed for their abilities to modulate ultraviolet (UV)-induced mutagenesis in Escherichia coli WP2. In addition to the previously reported comutagenic effect of arsenite, salts of three other compounds were found to enhance UV mutagenesis. CuCl2, MnCl2 (and a small effect by KMnO4), and NaMoO4 acted as comutagens in E coli WP2, which has wild-type DNA repair capability, but were much less comutagenic in the repair deficient strain WP2s (uvrA). The survival of irradiated or unirradiated cells was not affected by these compounds. No effects on UV mutagenesis were seen for 16 other metal compounds. We suggest that the comutagenic effects might occur either via metal-induced decreases in the fidelity of repair replication or (in the case of CuCl2) via metal-induced depurination.

Comparison of in vivo and in vitro cytogenetic assay results.

Abstract: In vitro mutagenicity assays have largely replaced whole animal studies for screening compounds for genotoxic potential. While numerous comparisons have been made between the results of these assays and cancer assays in rodents, comparisons between in vitro and in vivo mutagenicity studies where the genetic endpoints are the same have not been published. To this extent, the published literature was reviewed for chemicals that had been tested in both in vitro and in vivo cytogenetic assays. Two hundred sixteen chemicals were identified, and definitive test results were obtained for 181 of them. Results from the two assays agreed on 126 of the compounds and of the 55 compounds for which the results did not agree, 53 were positive in vitro and negative in vivo. The proportion of "false positives" and the significance of the two "false negatives" are discussed.

Chemical enhancement of SA7 virus transformation of hamster embryo cells: evaluation by interlaboratory testing of diverse chemicals.

Abstract: Twelve chemicals from diverse structural classes were tested under code for their capacity to enhance the transformation of Syrian hamster embryo cells by simian adenovirus SA7 in two independent laboratories. Pretreatment of hamster cells with eight of those chemicals (reserpine, dichlorvos, methapyrilene hydrochloride, benzidine dihydrochloride, diphenylhydantoin, cinnamyl anthranilate, 11-aminoundecanoic acid, and 4,4'-oxydianiline) produced repeatable enhancement of SA7 transformation at two or more consecutive dose levels, which constitutes clear evidence of enhancing activity in this assay. Both toxic and nontoxic doses of each of these chemicals caused enhancement of virus transformation. Two chemicals (2,6-dichloro-p-phenylenediamine and cinnamaldehyde) produced some evidence of enhancing activity (repeatable transformation enhancement at one dose). Dose ranges for cytotoxicity and enhancement of SA7 transformation were similar in both laboratories for all chemicals producing activity. The final two chemicals, chloramphenicol sodium succinate and ethylene thiourea, failed to reproducibly demonstrate either significant cytotoxicity or enhancement of SA7 transformation at concentrations up to 10-20 mM. The test results for these 12 chemicals were combined with the test results for 9 known carcinogens and noncarcinogens in order to evaluate relationships between activity, dose response, and lowest effective enhancing concentration for these compounds, as well as to correlate them with rodent carcinogenesis classifications. The Syrian hamster embryo cell-SA7 system demonstrated reproducible test responses in both intra- and interlaboratory studies and detected 13 out of 15 known rodent carcinogens.

Tests for mutagenic effects of ammoniated glycyrrhizin, butylated hydroxytoluene, and gum arabic in rodent germ cells

Abstract: Ammoniated glycyrrhizin, butylated hydroxytoluene, and gum Arabic are "generally recognized as safe" (GRAS) substances that are used primarily as additives in foods. These substances were incorporated into rodent diets and fed to male rats and mice for 10 and 8 wk, respectively. The treated male mice and rats were then tested for dominant lethal effects. The mice were also tested for induced heritable translocation. Results of the rat studies indicated a statistically significant dominant lethal effect of each of the compounds tested; however, the biological significance of this response is not known. Results of the mouse dominant lethal and heritable translocation studies, on the other hand, indicated no adverse effects of the compounds tested.

Comparison of S9 mix and hepatocytes as external metabolizing systems in mammalian cell cultures: cytogenetic effects of 7,12-dimethylbenzanthracene and aflatoxin B1.

Abstract: Two external metabolizing systems, S9 mix from Aroclor-induced rat livers and freshly isolated hepatocytes, were used for activation in cultures of human lymphocytes and V79 cells. 7, 12-dimethylbenzanthracene (DMBA) and aflatoxin B1 (AFB1) were employed as indirectly acting reference mutagens. Mutagenic effects were measured by induction of sister chromatid exchange (SCE). With DMBA, SCE-inducing effects were found to be quite similar after activation by S9 mix and activation by hepatocytes. In human lymphocytes nearly the same dose-effect relationships were found with both metabolizing systems; in V79 cells the hepatocyte-mediated induction of SCE was detectable at slightly lower concentrations than the S9-mediated SCE induction. In contrast with AFB1, S9 activation led to a stronger SCE induction than hepatocyte activation in both target cells. The induction of chromosomal aberrations by AFB1 after activation by the two metabolizing systems was also analysed in V79 cells. This experiment again revealed that AFB1 was more efficiently activated by S9 mix than by hepatocytes, and it appeared that AFB1 is a more potent inducer of chromosomal aberrations than of SCE. The different activation capacities of the two metabolizing systems for AFB1 may be due to the maintenance of inactivation mechanisms in hepatocytes or to the Aroclor induction of the S9 fraction. Our experiments have shown that the suitability of hepatocytes as an activation system is not restricted to microbial or eukaryotic point mutation assays, but that hepatocyte metabolism can also be successfully included in cytogenetic tests with short- and long-term cultures of mammalian target cells.