Enhancement of goldfish virus-2 in vitro replication by the pesticides carbaryl and toxaphene.

Goldfish virus-2 replication was enhanced in vitro by pretreatment of CAR cells with subcytotoxic concentrations of carbaryl and toxaphene. This phenomenon was time and temperature dependent. Shortening of pretreatment with carbaryl eliminated enhancement, which was observed for toxaphene only with substantially increased concentrations. Decreasing the temperature of pretreatment (4 degrees C) abrogated any enhancement by carbaryl and resulted in enhancement by toxaphene only at increased concentrations. Increased absorption of input virus was ruled out as a mechanism for enhancement, as was stimulation of cell division in the presence of pesticides over that of control cultures. Pretreatment of virus rather than cells did not result in enhancement.     

Characterization of varicella-zoster virus enhancement by the pesticide carbaryl.

Further characterization of the viral enhancement system of varicella-zoster virus by the pesticide carbaryl (1-naphthyl-N-methylcarbamate) is presented. It was necessary to expose cells to the enhancing chemical during the period of virus replication to detect enhancement. The optimum time for the pretreatment is 20 to 24 h. Maximum enhancement of virus expression occurs 48 to 72 h post-inoculation. Treated cells cannot pass on to daughter cells the ability to produce increased amounts of virus. alpha-Naphthol, a metabolite of carbaryl, is also capable of enhancing virus replication; Guthion, another pesticide tested, did not enhance varicella-zoster virus. The stable cell line HEP-2 can be used in place of human embryonic lung cells to detect enhancement. Differences in enhancement levels were not due to cell lot, cell passage, or a change in the stability of the cell membrane to sonic disruption.     

Characterization of varicella-zoster virus enhancement by the pesticide carbaryl

Further characterization of the viral enhancement system of varicella-zoster virus by the pesticide carbaryl (1-naphthyl-N-methylcarbamate) is presented. It was necessary to expose cells to the enhancing chemical during the period of virus replication to detect enhancement. The optimum time for the pretreatment is 20 to 24 h. Maximum enhancement of virus expression occurs 48 to 72 h post-inoculation. Treated cells cannot pass on to daughter cells the ability to produce increased amounts of virus. alpha-Naphthol, a metabolite of carbaryl, is also capable of enhancing virus replication; Guthion, another pesticide tested, did not enhance varicella-zoster virus. The stable cell line HEP-2 can be used in place of human embryonic lung cells to detect enhancement. Differences in enhancement levels were not due to cell lot, cell passage, or a change in the stability of the cell membrane to sonic disruption.     

Enhancement of varicella-zoster virus replication in cultured human embryonic lung cells treated with the pesticide carbaryl

In studies designed to determine the factors responsible for control of herpesvirus replicaton in an infected cell, we examined the interaction of varicella-zoster (VZ) virus-infected human embryonic lung cells with the pesticide carbaryl. The replication of the cell-associated VZ virus was enhanced 2- to 13-fold as compared to control cultures in Sevin 4 Oil-treated cultures and in cultures treated with the pesticide's active ingredient, carbaryl. The replication of VZ virus in cultures teated with the base oil plus inert ingredients found in the pesticide formulation was not enhanced. Possible differences in cytotoxicity induced by Seven 4 Oil, pure carbaryl, or the base oil preparation were ruled out since treated and control cultures were shown to have similar numbers of viable cells when measured by trypan blue exclusion tests or by the ability of treated cells to form foci. A dose response study showed a decrease in viral enhancement in cells treated with decreasing carbaryl concentrations.     

Enhancement of varicella-zoster virus replication in cultured human embryonic lung cells treated with the pesticide carbaryl.

In studies designed to determine the factors responsible for control of herpesvirus replicaton in an infected cell, we examined the interaction of varicella-zoster (VZ) virus-infected human embryonic lung cells with the pesticide carbaryl. The replication of the cell-associated VZ virus was enhanced 2- to 13-fold as compared to control cultures in Sevin 4 Oil-treated cultures and in cultures treated with the pesticide's active ingredient, carbaryl. The replication of VZ virus in cultures teated with the base oil plus inert ingredients found in the pesticide formulation was not enhanced. Possible differences in cytotoxicity induced by Seven 4 Oil, pure carbaryl, or the base oil preparation were ruled out since treated and control cultures were shown to have similar numbers of viable cells when measured by trypan blue exclusion tests or by the ability of treated cells to form foci. A dose response study showed a decrease in viral enhancement in cells treated with decreasing carbaryl concentrations.     

Variation in human herpesvirus susceptibility to enhancement by the pesticide carbaryl.

The ability of various human herpesviruses to be enhanced by the pretreatment of human embryonic lung cells with the pesticide carbaryl (1-naphthyl-N-methyl-carbamate) differs according to the virus tested. Different strains of varicella-zoster virus produced different patterns of susceptibility to enhancement. Laboratory-adapted strains were less sensitive to enhancement than were wild-type strains recently isolated from clinical specimens. The related human herpes simplex viruses types 1 and 2 and cytomegalovirus were negative for susceptibility to enhancement when either laboratory-adapted or wild-type strains were tested. No difference in the pattern of susceptibility was detected whether virus yields were determined by cell-associated or cell-free virus assay or when the input multiplicity was varied 10-fold.     

Variation in human herpesvirus susceptibility to enhancement by the pesticide carbaryl

The ability of various human herpesviruses to be enhanced by the pretreatment of human embryonic lung cells with the pesticide carbaryl (1-naphthyl-N-methyl-carbamate) differs according to the virus tested. Different strains of varicella-zoster virus produced different patterns of susceptibility to enhancement. Laboratory-adapted strains were less sensitive to enhancement than were wild-type strains recently isolated from clinical specimens. The related human herpes simplex viruses types 1 and 2 and cytomegalovirus were negative for susceptibility to enhancement when either laboratory-adapted or wild-type strains were tested. No difference in the pattern of susceptibility was detected whether virus yields were determined by cell-associated or cell-free virus assay or when the input multiplicity was varied 10-fold.     

Suppression of interferon synthesis by the pesticide carbaryl as a mechanism for enhancement of goldfish virus-2 replication

Interferon production was demonstrated by the goldfish-derived CAR cell line in response to infection by goldfish virus-2. Supernatants of infected cultures provided antiviral protection to CAR cells and another cell line derived from goldfish, ABIII. The protective factor retained activity after ultracentrifugation, dialysis, freezing and thawing, acid treatment (pH 2), or heating to 56 degrees C but was sensitive to trypsin. Supernatants of infected cultures did not affect adsorption of virus. Previous studies have shown that replication of goldfish virus type 2 is enhanced by pretreatment of cultures with subcytotoxic concentrations of carbaryl. In the present study, pesticide-treated cultures were found to synthesize reduced levels of interferon.     

Suppression of interferon synthesis by the pesticide carbaryl as a mechanism for enhancement of goldfish virus-2 replication.

Interferon production was demonstrated by the goldfish-derived CAR cell line in response to infection by goldfish virus-2. Supernatants of infected cultures provided antiviral protection to CAR cells and another cell line derived from goldfish, ABIII. The protective factor retained activity after ultracentrifugation, dialysis, freezing and thawing, acid treatment (pH 2), or heating to 56 degrees C but was sensitive to trypsin. Supernatants of infected cultures did not affect adsorption of virus. Previous studies have shown that replication of goldfish virus type 2 is enhanced by pretreatment of cultures with subcytotoxic concentrations of carbaryl. In the present study, pesticide-treated cultures were found to synthesize reduced levels of interferon.     

Spindle disturbances in mammalian cells. I. changes in the quantity of free sulfhydryl groups in relation to survival and c-mitosis in V79 chinese hamster cells after treatment with colcemid,diamide, carbaryl and methyl mercury

Asynchronously growing V79 Chinese hamster cells were treated with colcemid, diamide, carbaryl and methyl mercury, which are all known to be spindle disturbing agents. For each compound the dose response for c-mitosis, survival and level of free sulfhydryl groups was investigated under comparable conditions. Diamide, carbaryl and methyl were all found to give a significant increase of c-mitosis at a dose giving a decrease of non-protein sulfhydryl groups (NPSH, mainly glutathione) of 30–40% suggesting that a decrease of this magnitude may have a predictive value for spindle disturbances. Despite this similarity at concentrations close to the respective thresholds it was found that the c-mitotic activity at higher concentrations was not a simple function of average NPSH decrease. Diamide, which rapidly oxidizes glutathione to glutathione disulfide, was a less efficient c-mitotic agent than carbaryl and methyl mercury in relation to average NPSH decrease at higher concentrations. Protein bound sulfhydryl groups (PSH) were not significantly affected with diamide and carbaryl at their lowest c-mitotic concentrations while methyl mercury caused a significant decrease already at concentrations below the lowest c-mitotic concentration. With colcemid a significant decrease of average NPSH (14%) and PSH (12%) was observed only with concentrations giving close to 100% c-mitotic cells. Concentrations giving more than 20% c-mitosis gave a pronounced decrease of survival with carbaryl, diamide and methyl mercury while no toxic effects were obtained with colcemid, not even with concentrations giving close to 100% c-mitosis. Carbaryl, diamide and methyl mercury caused increased glutathione peroxidase activity indicating that these compounds cause increased lipid peroxidation. The possible connection between peroxidative damage of membranes and c-mitosis is discussed.     

Activation of vesicular stomatitis virus fusion with cells by pretreatment at low pH

Fusion of vesicular stomatitis virus (VSV) with Vero cells was measured after exposure of the virus to low pH under a variety of experimental conditions. The method of relief of fluorescence self-quenching of the probe octadecylrhodamine was used to monitor fusion. Incubation of the virus at pH 5.5 prior to binding to cells led to significant enhancement of fusion at the plasma membrane, whereas fusion via the endocytic pathway was inhibited. Fusion of pH 5.5-pretreated VSV showed a similar pH threshold for fusion as nontreated virus, and it was blocked by antibody to VSV G protein. Activation of VSV by pretreatment at low pH was only slightly dependent on temperature. In contrast, when VSV was first bound to target cells and subsequently exposed at 4 degrees C to the low pH, activation of the fusion process did not occur. The pH 5.5-mediated activation of VSV could be reversed by returning the pH to neutral in the absence of target membranes. The low pH pretreatment also led to aggregation of virus; large aggregates could be pelleted by low speed centrifugation and only the effects of the supernatant, which consist of single virions and/or microaggregates, were considered. The data were analyzed in the framework of an allosteric model according to which viral spike glycoproteins undergo a pH-dependent conformational transition to an active (fusion-competent) state. Based on that analysis we conclude that the conformational transition to the active state is rate-limiting for fusion and that the viral spike glycoproteins are fusion-competent only in their protonated form.     

The action of carbaryl and its metabolite alpha-naphthol on mitosis in V79 Chinese hamster fibroblasts. Indications of the involvement of some cholinester in cell division

Carbaryl (1-naphthyl-N-methylcarbamate) is a spindle-disturbing agent. A number of effects probably contribute to this activity. Carbaryl efficiently lowers the intracellular level of glutathione, thus endangering the integrity of the spindle. Carbaryl also causes lipid peroxidation. With anti-oxidant pretreatment, the frequency of c-mitotic cells is lowered which indicates that lipid peroxidation is a partial cause of the spindle-disturbing activity. At high concentrations of carbaryl, monopolar configurations in combination with cleavage are frequent and when tested, alpha-naphthol, which is thought to be formed from carbaryl in significant amounts at these concentrations, also gave monopolar configurations but with significantly lower frequencies of concomitant cleavage. Carbaryl inhibits cholinesterases and when tested, another cholinesterase inhibitor, diisopropylfluorophosphate, in combination with alpha-naphthol also increased the frequency of monopolar configuration in combination with cleavage. We therefore propose the involvement of some cholinester in the process of cell division.     

Genotoxic activity of a technical toxaphene mixture and its photodegradation products in SOS genotoxicity tests

Toxaphene (CAS No. 800-35-2) is a complex mixture of several hundred components that was used worldwide primarily as an agricultural pesticide with insecticide effects in the second half of the 20th century. In vitro investigations of the genotoxicity and mutagenicity of toxaphene were generally described in the literature, but they provided somewhat equivocal results. We re-evaluated the genotoxicity of technical toxaphene in two prokaryotic systems. The SOS Chromotest showed high sensitivity to toxaphene: three concentrations (40, 20 and 10 mg/l) were clearly positive and the dose-response effect was evident. In the umuC assay, a dose-dependent increase in genotoxic activity was observed at toxaphene concentrations from 2.5 to 40.0 mg/l, but these results were found to be not significant. The genotoxicity of toxaphene and its photodegradation products after UV-irradiation (3-6-9 h) at concentrations ranging from 7.5 to 60.0 mg/l was also examined in this study. An irradiated solution of technical toxaphene after 3 h showed no significant evidence of bacterial growth inhibition. However, exposure of Salmonella to 6 h UV-irradiated toxaphene showed a toxic effect compared with the negative control. After 9 h irradiation, a decrease of bacterial growth was observed. Activity of beta-galactosidase in the presence of a toxaphene solution was significantly increased after 6 and 9 h irradiation, reaching values that were 2.4- and 3.1-fold higher, respectively, than the control, which exceeded the criteria of significant genotoxicity. These results show that while technical toxaphene is a weak, direct-acting mutagen in some bacterial tests, a dose-dependent toxicity and genotoxicity of its photoproducts could be conclusively demonstrated by the umuC test.     

Nutrient enrichment in the High Arctic associated with Thule Inuit whalers: a paleolimnological investigation from Ellesmere Island (Nunavut,Canada)

The combined influence of a pesticide (carbaryl) and a cyanotoxin (microcystin LR) on the life history of Daphnia pulicaria was investigated. At the beginning of the experiments animals were pulse exposed to carbaryl for 24 h and microcystins were delivered bound in Microcystis’ cells at different, sub-lethal concentrations (chronic exposure). In order to determine the actual carbaryl concentrations in the water LC–MS/MS was used. For analyses of the cyanotoxin concentration in Daphnia’s body enzyme-linked immunosorbent assay (ELISA) was used. Individual daphnids were cultured in a flow-through system under constant light (16 h of light: 8 h of dark), temperature (20°C), and food conditions (Scenedesmus obliquus, 1 mg of C l⁻¹). The results showed that in the treatments with carbaryl egg numbers per female did not differ significantly from controls, but the mortality of newborns increased significantly. Increasing microcystin concentrations significantly delayed maturation, reduced size at first reproduction, number of eggs, and newborns. The interaction between carbaryl and Microcystis was highly significant. Animals matured later and at a smaller size than in controls. The number of eggs per female was reduced as well. Moreover, combined stressors caused frequent premature delivery of offspring with body deformations such as dented carapax or an undeveloped heart. This effect is concluded to be synergistic and could not be predicted from the effects of the single stressors.     

SCE induction and cell-cycle delay by toxaphene

Toxaphene is genotoxic in mammalian cell systems and also inhibits cell replication. It was therefore used to investigate possible masking of SCE induction due to cell-cycle delay. In this study, toxaphene-treated Chinese hamster lung (Don) cells exhibited a dose-dependent decrease in cell-cycle progression compared with untreated cells. At high, nontoxic toxaphene levels (15 micrograms/ml), cell cycling also slowed as the toxaphene treatment time was increased. Toxaphene induced significantly higher numbers of SCEs in treated cells, demonstrating a dose- and treatment time-relationship. Slopes of dose-response curves were 0.29, 0.43 and 0.77 SCE/micrograms toxaphene for 20.5 h, 24.5 h and 28.5 h incubation, respectively. There were no changes in SCE values in control cultures even when slower dividing cells were sampled e.g. at longer incubation times. Thus, higher SCE values in Chinese hamster cells were not associated per se with slower or more delayed cells. The results demonstrate that longer toxaphene treatment times were not necessary for obtaining sufficient harlequin-stained cells for SCE analysis, but that higher numbers of SCEs occurred in slower dividing cells, following prolonged incubation of cultures treated with toxaphene.     

Genotoxic activity of a technical toxaphene mixture and its photodegradation products in SOS genotoxicity tests

Toxaphene (CAS No. 800-35-2) is a complex mixture of several hundred components that was used worldwide primarily as an agricultural pesticide with insecticide effects in the second half of the 20th century. In vitro investigations of the genotoxicity and mutagenicity of toxaphene were generally described in the literature, but they provided somewhat equivocal results. We re-evaluated the genotoxicity of technical toxaphene in two prokaryotic systems. The SOS Chromotest showed high sensitivity to toxaphene: three concentrations (40, 20 and 10 mg/l) were clearly positive and the dose–response effect was evident. In the umuC assay, a dose-dependent increase in genotoxic activity was observed at toxaphene concentrations from 2.5 to 40.0 mg/l, but these results were found to be not significant. The genotoxicity of toxaphene and its photodegradation products after UV-irradiation (3–6–9 h) at concentrations ranging from 7.5 to 60.0 mg/l was also examined in this study. An irradiated solution of technical toxaphene after 3 h showed no significant evidence of bacterial growth inhibition. However, exposure of Salmonella to 6 h UV-irradiated toxaphene showed a toxic effect compared with the negative control. After 9 h irradiation, a decrease of bacterial growth was observed. Activity of β-galactosidase in the presence of a toxaphene solution was significantly increased after 6 and 9 h irradiation, reaching values that were 2.4- and 3.1-fold higher, respectively, than the control, which exceeded the criteria of significant genotoxicity. These results show that while technical toxaphene is a weak, direct-acting mutagen in some bacterial tests, a dose-dependent toxicity and genotoxicity of its photoproducts could be conclusively demonstrated by the umuC test.     

Biochemical transformation of mouse cells by herpes simplex virus type 2: enhancement by means of low-level photodynamic treatment.

The biochemical transformation of thymidine kinase-deficient cells by UV-inactivated herpes simplex virus is enhanced by low-level photodynamic treatment of the infected cells. At the concentration of proflavine used, the virus was not inactivated and both virus and cellular DNA syntheses were only marginally inhibited. The observed enhancement of the transfer of a virus gene to the cell genome suggests a possible cocarcinogenic role for photodynamically active dyes at very low concentrations.     

Sister-chromatid exchanges and thioguanine resistance in V79 Chinese hamster cells after treatment with the aneuploidy-inducing agent carbaryl +/- S9 mix

Carbaryl induced sister-chromatid exchanges (SCEs) but no thioguanine resistance in V79 Chinese hamster cells. Addition of S9 from Aroclor-pretreated rats, or glutathione, reduced the toxic effects of carbaryl. Glutathione or S9 mix reduced the effect of carbaryl on SCE. However, the latter result indicates that carbaryl's effect may be enhanced at a certain compound/S9 ratio. Since treatment with microsomes alone, but not S9 mix, was clastogenic it cannot be excluded that this enhancement of SCE was due to perturbations in the S9 mix by carbaryl rather than to formation of some particular SCE-inducing metabolite from the compound. The effects of carbaryl on chromosomes and chromosomal distribution are comparable to those sometimes reported for TPA. This, in conjunction with the weak indications on carcinogenic activity of carbaryl, makes it of interest that the compound be tested for promotion or co-carcinogenicity in vivo.     

Spindle disturbances in mammalian cells. II. Induction of viable aneuploid/polyploid cells and multiple chromatid exchanges after treatment of V79 Chinese hamster cells with carbaryl. Modifying effects of glutathione and S9

Treatment of V79 cells with carbaryl increased the frequency of cells with elevated chromosome numbers (greater than 22). This effect of carbaryl was inhibited by simultaneous addition of glutathione or S9. Although selective forces seemed to act against cells with increased chromosome numbers, such cells were still significantly more frequent in treated compared with control cultures 74 h after treatment. In addition, a high adaptive value for these remaining aneuploid/polyploid cells was indicated because they were able to go through 2 successive rounds of replication immediately before fixation to the same extent as cells with chromosome numbers considered normal (21/22) for this cell line. Multiple chromatid exchanges were also observed after carbaryl treatment. The lack of single exchange events indicates that the effect may be systemic. However, additional experiments are required before this hypothesis can be confirmed or discarded. Considering the results obtained and the possible importance of gene dosage for tumor promotion, it is suggested that carbaryl should be tested for tumor promotion in vivo.