Biotesting of wastewater: a comparative study using the Salmonella and CHO assay systems.

Means to assess the toxicity of wastewaters are essential to implementing the Federal Clean Water Act. Health risk assessment based on single chemicals is limited by the number of chemicals that can be identified and to those chemicals for which toxicity data are available. Long-term whole animal tests on large numbers of wastewater samples are not practical. In this study, two short-term tests, the Salmonella mutagenicity assay and the Chinese hamster ovary (CHO) cell assay for mutagenicity and cytotoxicity, were evaluated as potentially useful biomonitors of wastewaters. Standard assay protocols were modified to allow testing of up to 2.5 and 3.4 ml of unconcentrated water in the bacterial and mammalian cell tests, respectively. Cytotoxicity and mutagenicity were detected in some unconcentrated wastewater samples using these modifications. Data on eight wastewater samples, representing five different sites, indicated that the Salmonella test is the more sensitive indicator of mutagenic activity in those samples, whereas the CHO test is a sensitive indicator of the presence of cytotoxic components. Wastewater concentrates, prepared by adsorption onto XAD-2 and "blue cotton," were compared in the two bioassays. In a single concentrate, the two short-term tests detected distinctly different mutagens. Advantages of using the CHO-AS52 cell line instead of the CHO-K1BH4 line for detecting wastewater mutagens were indicated. This study illustrates the complementary use of multiple bioassays and concentration methods to detect and characterize toxic components in wastewater.

Multiple-endpoint mutagenesis with Chinese hamster ovary (CHO) cells: evaluation with eight carcinogenic and non-carcinogenic compounds.

Previously, we have shown that Chinese hamster ovary (CHO) cells are useful for quantifying chemical-induced gene mutations. We have defined the conditions of a Multiplex CHO System which permits determination of mutagen-induced chromosome aberration, and sister chromatid exchange (SCE) in addition to cytotoxicity and gene mutation in the same treated culture. This allows us to extend the spectrum of quantitative mutagenesis to include clastogenic endpoints. In the present study, we used four carcinogenic/noncarcinogenic pairs to validate the relative utility and sensitivity of each endpoint, and to study the interrelationship of these four distinct biological effects. These compounds include the direct-acting carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ICR 170 and their noncarcinogenic analogue N-methyl-N'-nitroguanidine (MNG) and ICR 170-OH, and the procarcinogens benzo[a]pyrene (B[a]P) and dimethylnitrosamine (DMN) and their noncarcinogenic analogues pyrene and dimethylamine (DMA) respectively. A rat liver homogenate preparation (S9) was used to assay for the biological activities of procarcinogens. Under our experimental conditions, we observed that carcinogens DMN, B[a]P, MNNG and ICR 170, but not their noncarcinogenic counterparts, showed all four biological effects. Our studies with these chemicals showed that cytotoxicity does not necessarily correlate with any of the genetic endpoints. On a molar basis, noncarcinogens, pyrene and ICR 170-OH show similar toxicity to carcinogens B[a]P and ICR 170, respectively. The other two non-carcinogenic analogues, DMA and MNG, exhibit minimal toxicity at concentrations 10-1,000 times higher than cytotoxic concentrations of the corresponding carcinogens, DMN and MNNG. In general, gene mutation and SCE are more sensitive than chromosome aberration assay. The gene mutation assay is more specific than SCE and chromosome aberration assays since none of the noncarcinogens exhibit a detectable response in the gene mutational assay. ICR 170 and MNNG are much more active than B[a]P and DMN as ranked on a molar basis. These results indicate that the Multiplex CHO System is capable of discriminating divergent structural classes of carcinogenic and noncarcinogenic compounds, such as the eight chemicals chosen for our study.

Evidence for reactive oxygen species inducing mutations in mammalian cells.

We have studied the mutagenicity (by selecting for mutants resistant to 6-thioguanine) and cytotoxicity (by determining cellular cloning efficiency) of physical and chemical agents in Chinese hamster ovary (CHO) cells, clone CHO-K1-BH4 (K1-BH4), and its radiation-hypersensitive transformant, AS52. AS52 cells contain a single functional copy of a bacterial gene, the xanthine/guanine phosphoribosyltransferase (gpt) gene instead of its mammalian equivalent, the hypoxanthine/guanine phosphoribosyltransferase (hprt) gene. We found that x-ray and neutron irradiations are equally toxic to both cell types; however, these physical agents are approximately equal to 10 times more mutagenic to AS52 cells than to K1-BH4 cells. Our earlier studies using Southern blot analysis showed that x-irradiation produces mostly or exclusively deletion mutations in both cell types. If reactive oxygen species mediate the mutagenic effects of radiations and chemicals, then radiomimetic compounds such as streptonigrin and bleomycin, which exert their biological effects via reactive oxygen species, and oxidizing compounds such as potassium superoxide and hydrogen peroxide should elicit a similar differential mutagenic response in both cell types. On the other hand, agents such as ethyl methanesulfonate, ICR 191, and UV light, which do not produce reactive oxygen species, should not elicit differential mutagenicity. Our results fulfill such predictions. The apparent hypermutability of AS52 cells probably results from a higher recovery of multilocus deletion mutants in AS52 cells than in K1-BH4 cells, rather than a higher yield of induced mutants.

The toxicity of sixteen metallic compounds in Chinese hamster ovary cells.

The toxicity of 16 metal salts to Chinese hamster ovary (CHO) cells was determined by measuring the cloning efficiency (CE) of CHO cells after exposure to the metals. CHO cells differed by a factor of 10(5) to 10(6) in their toxic response to these metal salts. While Cd(II) was the most toxic ion, Mg(II) exhibited the least toxicity based on either CE50 (concentration required to reduce the CE to 50%) or D0 (concentration increment which reduced the CE by 63%). On the basis of CE50, the toxicity ranking was Ag greater than Tl for monovalent metals, Cd greater than Zn greater than Hg greater than Co greater Cu greater than Mn greater than Ni greater than Be greater than Pd greater than Sr greater than Mg for divalent metals, and In greater than Rh greater than Y for trivalent metals. A similar ranking was found for D0. For the 11 divalent metals, correlations of CE50 and D0 in the CHO cell assay and the Pearson-Mawby softness parameter for metals (sigma p) were reasonably strong. A good correlation exists between the results of this study on the toxic response in CHO cells and published data on toxicity in mice and Drosophila. It appears that the CHO cell cloning assay may be useful in preliminary screening of metallic compounds as an indicator of their predicted toxicity in higher organisms.

Mutagenicity and cytotoxicity of dimethyl and monomethyl sulfates in the CHO/HGPRT system.

It has recently been shown that coal fly ash collected from coal-fired plants contains dimethyl sulfate (DMS) and monomethyl sulfate (MS) at concentrations as high as 830 ppm. Both these compounds were tested in the CHO/HGPRT system, and it was found that only DMS was cytotoxic and mutagenic to CHO cells. On a molar basis, DMS is twice a mutagenic as methyl methanesulfonate (MMS). Under our treatment conditions, maximum mutation induction and cytotoxicity were obtained after approximately 1 h. The Mutagenic potency of DMS was more stable in aqueous solutions at 4 degrees C than at the ambient temperature of 22 degrees C, but was least stable in DMSO solutions at 22 degrees C. Near-ultraviolet (near-UV) light caused an approximately twofold decrease in the mutagenic and cytotoxic effects of DMS. Although DMS produced by coal combustion could be rendered innocuous by environmental agents in a short span of time, this compound could still pose a health risk to workers closely involved in coal-combustion technology.

Utilization of Chinese hamster cells in vitro and in vivo in genetic toxicology: a multiphasic approach.

An approach often used to study genetic risk is comparative mutagenesis in different organisms. We have begun the development and validation of a multiphasic genetic toxicity system using the Chinese hamster and its somatic cells to measure mutational events in the same (or similar) gene, the same chromosome derived from the same animal (Fig. 1). This system will eliminate much of the uncertainty generated when different mutational events in such evolutionally divergent organisms as bacteria, insects, and mammals are compared. Using CHO cells we have defined an assay, CHO/HGPRT, to determine mutation at the hgprt locus. Coupled with S9 metabolic activation system, the CHO/HGPRT assay can quantify gene mutation and cytotoxicity induced by various classes of chemicals, physical agents, and the combination thereof. The quantitative nature of this assay permits elucidation of the structure-activity relationship for a given class of direct-acting agents. By incorporating the cytogenetics of CHO cells into this assay we can simultaneously measure induced chromosome aberrations and SCE. Using the stable CHO/human hybrid cell line AL-J1 measurement of chromosome deletion and/loss can be also performed. In order to further expand the usefulness of this genetic toxicity system to the molecular and whole animal levels we have begun development and validation of two additional systems. To study the molecular events which may result in mutation we are developing a CHOpSVgpt system. A Chinese hamster system with treatment in vivo is being developed to study mutation at the hgprt locus, chromosome aberration, and SCE in spleen cells in vitro. The use of this multiphasic genetic toxicity system at the cellular, molecular, and animal levels may soon provide reliable and rapid identification of suspected environmental mutagens.

Cytotoxicity and mutagenicity of alkylating agents in cultured mammalian cells (CHO/HGPRT system): mutagen treatment in the presence or absence of serum.

Cytotoxicity and mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT system) were measured for a range of concentrations of 6 alkylating agents [methyl and ethyl methanesulfonate (MMS, EMS), N-methyl- and N-ethyl-N'-nitro-N-nitrosoguanidine (MNNG, ENNG), and methyl- and ethyl-nitrosourea (MNU, ENU)] to determine the effect of the presence or absence of serum during the time of mutagen treatment. Cultures were treated with the mutagens for 5 h, a time period which results in no growth inhibition in the absence of serum, to estimate the potential decrease in effective mutagen dose to the cells which might result from reactivity with the serum proteins. With all 6 agents, identical results were found for cytotoxicity and for mutagenicity regardless of the presence or absence of serum during treatment. This finding demonstrates that the use of serum in cell-culture medium does not present any problems in apparent dosimetry studies, at least with these alkylating agents.

Effects of antipain (a protease inhibitor) on respiration, viability, and excision of pyrimidine dimers in UV-irradiated Escherichia coli cells.

The protease inhibitor antipain increases the effectiveness of UV irradiation on cessation of respiration and cell killing in Escherichia coli B/r cultures without affecting excision of pyrimidine dimers. The actions are similar to those caused by cyclic AMP in irradiated cultures.

Role of cyclic adenosine 3',5'-monophosphate on cessation of respiration in ultraviolet-irradiated Escherichia coli.

The addition of cyclic adenosine 3',5'-monophosphate (cAMP) to ultraviolet-irradiated Escherichia coli B/r cultures causes additional cells to cease respiring and to die. These effects of cAMP are greater on glucose-grown cells, where the effects of ultraviolet radiations alone are smaller and where the intracellular concentrations of cAMP are known to be lower.

Delayed ultraviolet light-induced cessation of respiration by inadequate aeration of Escherichia coli.

Inadequately aerated Escherichia coli B/r cultures did not shut their respiration off 60 min after ultraviolet light (52 M/m2 at 254 nm) as they did when well supplied with oxygen. Since cessation of respiaration is associated with cell death, the result suggested that oxygen toxicity by superoxide radicals generated by cell metabolism might be responsible for cell death. The specific activity of superoxide dismutase, which scavenges O2- radicals, increased twofold after 90 min of adequate aeration, but the specific activity of catalase remained constant. Respiration and viability of irradiated cells were affected not at all by the presence of superoxide dismutase and only slightly by the presence of catalase. Metal ions such as Mn2+ and Fe2+ inducers of superoxide dismutase, had no effect on respiration and viability. When irradiated cells were incubated under N2 for 90 min, the respiration, growth, and viability time-course responses were the same as for the cells not exposed to anareobiosis. We conclude that superoxide anions generated at the time of irradiation play no part in cessation delays the ultraviolet light-induced synthesis of proteins responsible for the irreversible cessation of respiration.

Centrifugal separation of irradiated cultures of Escherichia coli cells into viable and nonviable populations.

Incubation of ultraviolet-irradiated Escherichia coli B/r cultures with 0.7% Triton X-100 resulted in a large decrease in turbidity. Under phase-contrast optics, most of the irradiated detergent-treated cells were smaller than normal and of low phase density; only a small percentage were normal or larger than normal and of normal phase density. Irradiated cells not treated with detergent showed fewer pronounced morphological changes. Irradiated cells treated with detergent lost large amounts of proteins and ribonucleic acid, but not of deoxyribonucleic acid. Such cultures could be separated by centrifugation into populations of (i) slowly sedimenting cells consisting of small, phase-light cells of low viability and (ii) large cells of normal phase density and high viability (100%). A similar separation was effected in gamma-irradiated cultures.

Pyrimidine dimer excision in surviving and nonsurviving cells of ultraviolet-irradiated cultures of Escherichia coli.

We compared dimer excision in viable and nonviable cells fractions separated from Escherichia coli B/r cultures exposed to ultraviolet (UV) irradiation. For cells grown on minimal medium with glycerol as a carbon source, both fractions from the irradiated (20 J/m2, 5% survival) culture excised 60 to 70% of the thymine dimers from prelabeled DNA within 120 min. This percentage was, within experimental error, the same as that obtained from unseparated cultures. When isolated viable and nonviable populations were given a second UV exposure (20 J/m2) both types of cells were again able to excise dimers. The UV survival curve for the isolated viable population indicates that these cells are no more sensitive to radiation than exponentially growing cells not previously exposed to UV. The extent of dimer excision after UV irradiation was also the same in viable and nonviable cells separated from cultures grown on a glucose minimal medium in which both populations excised about 85% of the dimers within 120 min. These results show that the extent of removal of pyrimidine dimer from deoxyribonucleic acid is not precisely correlated with survival of repair-competent bacterial cells after exposure to UV light.

Evidence relating cessation of respiration, cell envelope changes, and death in ultraviolet-irradiated Escherichia coli B-r cells.

Ionic and nonionic detergents have little effect on respiring bacteria, but in cultures poisoned with KCN rapid solubilization of the cell membrane, as indicated by turbidity losses, takes place. Ultraviolet radiations cause Escherichia coli cells grown in minimal medium with glycerol as a carbon source to cease respiring and growing about 1 h after irradiation. We tested the effect of the nonionic detergent Triton X-100 on growth and cell membrane dissolution (both measured by turbidity changes), respiration, and viability of unirradiated and irradiated E. coli B/r cells. When the detergent was added to cells immediately after irradiation, a decrease in turbidity occurred only when respiration was about to cease; when it was added after cessation of respiration, the turbidity loss was immediate. In both cases the turbidity loss was about 60%, and disintegration of the cell walls did not take place. 5-Fluorouracil (FU) and thermal (42 C) treatments cause respiration of irradiated cells to be maintained and also cause viability increases. Irradiated cells treated with FU and detergent show no turbidity loss just prior to the time respiration normally ceases, but a loss does occur in irradiated cells incubated with detergent at 42 C. We conclude that FU maintains respiration for all of the cells, but that thermal treatment maintains respiration for only part of the cells. In all cases the detergent had only a negligible effect on the respiration and viability of unirradiated and irradiated cells. We conclude that Triton X-100 causes solubilization of cell membranes of only nonrespiring cells that are not destined to survive.