Insect virus: assays for toxic effects and transformation potential in mammalian cells.

The nuclear polyhedrosis virus of Autographa californica (AcNPV) was evaluated by using in vitro test systems for toxicity and transforming potential in mammalian cells. Mass cell cultures of CV-1 and WI38 cells appeared unaffected by AcNPV at a multiplicity of infection of 5. Human foreskin cells grew more slowly after inoculation but eventually produced healthy monolayers. The sensitivities of the inhibition of reproductive survivability assays were greater and demonstrated slight AcNPV toxicity to CV-1, WI38, and human foreskin cells. Toxicity was not ameliorated when gradient-purified or psoralen-inactivated virus was used, suggesting that the toxic component of the preparation is part of the virion or copurifies with it. AcNPV was not toxic to and did not transform BALB/c 3T3 cells or primary cell cultures derived from Syrian hamster embryo cells (SHE). Unlike the BALB/c 3T3 transformation assay, the SHE assay detected no spontaneous transformants. The SHE transformation assay can employ simian adenovirus 7 as a positive control. SHE are transformed by numerous viruses and so are useful in assessment protocols. This study suggests that in vitro assessment of viral pesticide toxicity should employ the inhibition of reproductive survivability assay and that transformation assessment is best done with the SHE-simian adenovirus 7 procedure.

Relationships between benzo(a)pyrene-DNA adduct levels and genotoxic effects in mammalian cells.

The effectiveness of benzo(a)pyrene [B(a)P]-DNA binding as an internal dosimeter was evaluated. Data were obtained from concurrent studies, measuring B(a)P induced genotoxic effects and DNA adducts in several short-term bioassay systems: cytotoxicity, gene mutation, and sister chromatid exchange in Chinese hamster V79 cells; cytotoxicity, gene mutation, and chromosome aberrations in mouse lymphoma L5178Y TK+/-; cytotoxicity and enhanced virus transformation in Syrian hamster embryo cells; and cytotoxicity and morphological transformation in C3H10T1/2CL8 mouse embryo fibroblasts. Both total B(a)P-DNA binding and specific B(a)P-DNA adducts were measured. N2-(10 beta-[7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]yl)deoxyguanosine [BPDE I-dGuo] was one of the major adducts identified in all bioassay systems. DNA binding and genotoxic responses varied significantly between bioassays. Each genetic end point was induced with a differing efficiency on a per adduct basis. However, the relationships between frequency of genetic effect or morphological transformation and B(a)P-DNA binding or BPDE I-dGuo were linear within a given assay. In order to compare biological end points of diverse frequencies in diverse biological systems, a doubling adduct level, expressed as the number of BPDE I-dGuo adducts per unit of DNA required to double the induced frequency of biological response, was applied to the data.

Tumorigenesis and genotoxicity of ethyl carbamate and vinyl carbamate in rodent cells.

Vinyl carbamate (VC) is a suspect metabolic intermediate in ethyl carbamate (EC) carcinogenesis. In the present studies, EC and VC were evaluated for their relative abilities to induce adenomas and sister chromatid exchanges (SCEs) in lung cells of A/J, C3HeB/FeJ, and C57BL/6J strain mice. For both end points, animals were administered a single i.p. injection of the test chemical. Percentage of mice with adenomas and number of adenomas per mouse were compared among the three strains 24 weeks following exposure to EC or VC. Although the relative order of strain sensitivity was the same for both chemicals: A/J greater than C57BL/6J greater than C3HeB/FeJ, VC was much more potent than EC. For SCE analysis of primary lung cells cultured from treated animals, EC and VC showed potency differences similar to those observed for tumorigenesis. All three mouse strains revealed significant dose-dependent increases in SCE frequency. However, there was no strain specificity for this effect. SCE persistence over time was also compared in treated A/J and C57BL/6J mice. Although EC- and VC-induced SCE frequencies declined over a 2-week observation period, again, there was no strain specificity for this effect. VC was also tested for enhancement of SA7 virus transformation of Syrian hamster embryo cells. Significant concentration-dependent increases in cell transformation frequency were observed.

Analysis of the interlaboratory and intralaboratory reproducibility of the enhancement of simian adenovirus SA7 transformation of Syrian hamster embryo cells by model carcinogenic and noncarcinogenic compounds.

The intralaboratory and interlaboratory reproducibility of a DNA virus (SA7) transformation enhancement assay was investigated using nine carcinogenic and noncarcinogenic compounds representing a variety of chemical classes. By the use of standardized procedures designed to limit assay variables, replicate assay data were collected in two independent laboratories and analyzed for concurrence. The carcinogens, 7,12-dimethylbenz(a)anthracene, benzo(a)pyrene, and N-methyl-N'-nitro-N-nitrosoguanidine yielded reproducible dose-dependent cytotoxicity and positive transformation effects (defined as statistically significant [p less than or equal to 0.05] enhancement of virus transformation at two or more consecutive dose levels) in all experiments in both laboratories. The carcinogens lead chromate, diethylnitrosamine, 4-nitroquinoline-N-oxide, and 2-acetylaminofluorene demonstrated enhancement of SA7 transformation at two or more dose levels in 40-50% of the assays. The noncarcinogenic structural analogs anthracene and pyrene consistently did not produce positive assay responses when tested at dose levels up to the limits of solubility. Good interlaboratory concurrence was demonstrated for these model compounds in the Syrian hamster embryo cell-SA7 assay.

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

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.

Mutation and enhanced virus transformation of cultured hamster cells by exposure to gaseous ethylene oxide.

Ethylene oxide is a classical mutagen and a carcinogen based on evidence from studies in experimental animals. It is widely distributed in industrial, research, hospital, and food environments. In an effort to explore the use of newly developed methods for exposing mammalian cells to gaseous or volatile mutagens/carcinogens, Chinese hamster V79 cells were treated for 2 hr with gaseous ethylene oxide, in sealed treatment chambers, and assayed for survival and mutagenic response by analysis of induced resistance to 6-thioguanine or ouabain. Significant numbers of mutants were produced at both genetic markers by 1,250-7,500 ppm ethylene oxide. Similarly, primary Syrian hamster embryo cells were treated for 2 or 20 hr with gaseous ethylene oxide in sealed treatment chambers and subsequently assayed for survival and increased sensitivity to SA7 virus transformation. Treatment concentrations extended from toxic to several nontoxic concentrations. After 2-hr ethylene oxide treatment at 625-2,500 ppm a significant enhancement of virus transformation was observed. At 20 hr after treatment no enhancement was observed. Treatment of hamster cells with ethylene oxide in both bioassay systems yielded concentration-related, quantitative results.

Chemical enhancement of viral transformation in Syrian hamster embryo cells by gaseous and volatile chlorinated methanes and ethanes.

Methods were developed for exposing cells in vitro to gases or vapors of volatilized organic liquids. Compounds were selected for their industrial importance, environmental impact, and suspected role in the etiology of some human cancers. Exposure chambers were designed for easy insertion of dishes of cultured cells and were equipped with inlet and outlet ports for introduction and purging of test gases. A gas delivery system utilizing a mass flow meter was used for the quantitative distribution of test gases into exposure chambers. For volatile compounds, appropriate volumes of cold (4 degrees) liquids in glass Petri dishes were quickly placed into chambers, the system sealed, and the compounds rapidly volatilized at 37 degrees. For exposure, the cells and chambers were placed in an incubator and rocked at a constant rate so that a portion of the cells was always in direct contact with the test gases or vapors. Known sample volumes were removed after various treatment times and test gas concentrations determined by standard gas chromatographic techniques. After exposure, the cells were removed and assayed for viability and increased sensitivity to viral transformation. Under these experimental conditions, the volatile liquids 1,1,1-trichloroethane, dichloromethane, chloroform, 1,2-dichloroethane, and 1,1-dichloroethane significantly enhanced transformation of Syrian hamster embryo cells by SA7 adenovirus, while acetone exerted no effect. The gases chloromethane and vinyl chloride were also active in this test system, while bromomethane, methane, and ethane were inactive. Incorporation of some of these compounds into liquid cell culture medium for cell treatment was either unsuccessful or produced only a weak enhancement response. Methodology is now available to evaluate volatile and gaseous carcinogens or mutagens and can be used to identify their mechanisms of action and the relative hazards of these agents to human health.

Synergism in the transformation of hamster embryo cells treated with formaldehyde and adenovirus.

Formaldehyde is a large production volume chemical widely distributed in research laboratories, industrial workplaces, and home and personal environments. Inhalation studies with formaldehyde have documented its ability to produce squamous cell carcinomas in rats. When primary hamster embryo cells were treated by gaseous exposure to formaldehyde or by incorporation into the medium, a dose-related increase in the frequency of SA7 virus transformation was produced. The length of chemical treatment and the time interval before subsequent addition of transforming virus was critical, with two-hr treatment times as the most efficient. Treatment by gaseous exposure permitted utilization of lower treatment concentrations. Determination of formaldehyde concentrations in culture media of bioassay dishes treated by this method documented that 2.2 micrograms/ml produced significantly enhanced viral transformation. Exposure of hamster embryo cells to formaldehyde by these methods produces reproducible and quantitative genotoxic effects.

Enhancement of viral transformation of hamster embryo cells by pretreatment with 4-chloromethylbiphenyl.

Treatment of Syrian hamster embryo cells with 4-chloromethylbiphenyl produced reproducible and quantitative concentration-dependent enhancement of viral transformation the statistical significance of which was independent of cell lethality. These results suggest this compound has carcinogenic potential since positive results in this mammalian cell bioassay system closely correlate with the carcinogenic potential of many diverse compounds and with structurally similar agents, especially other chlorinated compounds.

Characterization of human cells transformed by chemical and physical carcinogens in vitro.

Several different classes of chemical carcinogens induced the transformation of human fibroblasts grown in vitro. Characteristics of the events that occur from time of treatment through the expression of neoplastic transformation are presented. The S-phase appeared to be the portion of the cell cycle most vulnerable to insult. Staging of the cells by blocking them in G1 before releasing them to proceed through scheduled DNA synthesis (S) was required to induce reproducible transformation. Compounds such as insulin were added to the cells upon release from the block to sensitize the cells to the carcinogen that was added during S. Growth of the transformed cells as distinct from nontransformed cells was promoted by growth in medium supplemented with 8X nonessential amino acids. Carcinogen-treated cells in the early stage of transformation exhibited abnormal colony morphology and were able to grow at 41 degrees C, in air atmosphere, and in medium supplemented with only 1% serum. In addition, the transformed cells were insensitive to KB cell lysate and exhibited density independent, as well as anchorage independent, growth (i.e., growth in 0.33% agar). Cells that grew in soft agar also produced undifferentiated mesenchymal tumors in preirradiated nude mice.

Characteristics of hamster cells transformed by the combined action of chemical and virus.

Pretreatment of hamster cells with chemical carcinogen enhances transformation by a simian adenovirus, SA7. Such transformants were compared to cells transformed by SA7 alone for the presence of virus-specified antigens, and for the ability to clone in soft agar and to form progressively growing sarcomas in hamsters. The SA7 "T" antigen content and the relative cloning efficiencies in agar of cell lines of both groups were similar. Some cell lines obtained by pretreatment with chemical carcinogen, however, produced a higher incidence of tumors, suggesting that the chemical carcinogen was involved in the transformation of cells by virus. A further indication that these cells differed from cells transformed by virus alone was obtained with MCA-transformed fibroblast cell lines that were subsequently also transformed in vitro by SA7 to cuboidal (SA7 type) morphology. These MCA-SA7-transformed cells produced a higher incidence of sarcomas than cells transformed by virus alone, but a lower tumor incidence than the parental MCA line.

RNA type C virus antigens in hamster cells transformed by carcinogenic DNA viruses and chemicals.

The passive hemagglutination inhibition technique was used to test serologically for the presence of Syrian hamster type C virus antigen(s) (SHCVA) in a wide variety of normal and transformed hamster cells and tissues. SHCVA could not be detected in normal tissues or nonneoplastic tissues of tumor-bearing Syrian hamsters. Normal hamster embryo cells or cells transformed in vitro by simian adenovirus, by chemical alone, or doubly transformed by simian adenovirus and chemical did not contain SHCVA; however, SHCVA was found in a majority of tumors resulting from transplantation of these in vitro-transformed cells. No consistent pattern was observed in the capacity of individual transformed cell lines to produce SHCVA-positive or -negative tumors. When cells of a given transformed line were inoculated at 4 sites on each of 8 hamsters, SHCVA-positive tumors were found not to be randomly distributed but rather to be clustered on a few animals. SHCVA could be detected in only a few primary tumors induced by inoculation of carcinogenic DNA viruses; however, both the incidence and titer of SHCVA were significantly increased in a variety of transplanted tumors. These data suggest that SHCVA may be introduced into transplanted, transformed hamster cell tumors during passage in the host animal. Alternatively, in vivo conditions may allow expression of viral antigens not found under in vitro conditions; however, if this is true, only certain animals appear to be capable of activating SHCVA.

Estradiol-induced wasting syndrome in conventionally reared and germ-free mice.

The pathogenesis of the estradiol-induced wasting syndrome is complex and cannot be explained solely by immunological impairment and subsequent infection.