Influence of aroclor 1254 on benzo(a)pyrene-induced DNA breakage, oxidative DNA damage, and cytochrome P4501A activity in human hepatoma cell line

Both polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) are important environmental pollutants. They coexist widely in the environment at very low levels. Numerous studies indicated that aroclor1254 (one of PCBs mixture) is the inducer of cytochrome P450 1A enzyme acitivity. Benzo(a)pyrene (BaP) can cause a variety of toxicities in vitro, such as oxidative DNA damage and genotoxicity. In the present study, HepG2 cells were treated with either BaP (50 microM) or aroclor1254 at concentrations of 11.5 (low), 23.0 (medium), and 46.0 microM (high) alone, or pretreated the cells with aroclor1254 (11.5, 23.0, and 46.0 microM), followed by BaP (50 microM). It was found that 7-ethoxyresorufin-O-deetylase (EROD) activities of HepG2 cells exposed to either BaP or aroclor 1254 increased. DNA damage measured by DNA migration and the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) also increased in cells exposed to BaP, but not in cells exposed to aroclor1254. Under the Aroclor 1254 pretreatment condition, BaP-induced EROD activities was enhanced in cells exposed to the medium and high concentrations of aroclor1254 (P < 0.01 for both), whereas in all pretreatment groups aroclor1254 significantly increased BaP-induced DNA migration (P < 0.01 for all) and the 8-OHdG formation (P < 0.05 for all). In addition, there was positive correlation between the EROD induction activity and Olive tail moment (r(2) = 0.958, P < 0.01) or the levels of 8-OHdG (r(2) = 0.992, P < 0.01). The findings suggest that under the experimental conditions aroclor1254 may enhance BaP-induced DNA migration and oxidative DNA damage in HepG2, due to inducing CYP1A enzyme activity.     

Vinclozolin, a widely used fungizide, enhanced BaP-induced micronucleus formation in human derived hepatoma cells by increasing CYP1A1 expression

Vinclozolin, a widely used fungicide, can be identified as a residue in numerous vegetable and fruit samples. To get insight in its genetic toxicity, we investigated the genotoxic effect of vinclozolin in the human derived hepatoma cell line HepG2 using the micronucleus (MN) assay. Additionally, to evaluate the co- or anti-mutagenic potency of vinclozolin, we treated HepG2 cells with different concentrations of vinclozolin for 24 h. Subsequently, the cells were exposed to benzo[a]pyrene (BaP) for 1h. Exposure of HepG2 cells to 50-400 microM vinclozolin alone did not cause any induction of micronuclei. However, a pronounced co-mutagenic effect was observed. MN frequencies caused by BaP increased by 30.6%, 52.8% and 65.3% after pretreatment of the cell cultures with 50, 100 and 200 microM vinclozolin, respectively. The highest concentration (400 microM) of vinclozolin tested caused cytotoxicity. Therefore, micronuclei were not considered for that concentration. To clarify the mechanism of cogenotoxicity, we assayed cytochrome P450 1A1 (CYP1A1), which plays a pivotal role in activation of BaP. Cells exposed to vinclozolin led to significant increase of CYP1A1 expression in Western blot. The result suggested that induction of CYP1A1 by vinclozolin account for its enhancing effect on genotoxicity caused by BaP.     

PCB126 enhanced the genotoxicity of BaP in HepG2 cells by modulating metabolic enzyme and DNA repair activities

Both of benzo(a)pyrene (BaP) and 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) are ubiquitous and persistent environmental pollutants. These two chemicals coexist in various environmental media and human samples and thus may have combined effects on human health. However, the toxic effects and related mechanism of co-exposure to BaP and PCB126 remain unknown. In a series of experiments using the HepG2 cells exposed to BaP (50 μM) or/and PCB126 (0.01, 0.1, 1 and 10 nM), we measured the rate of micronucleus (MN) formation, CYP1A1 activity and expression of nucleotide excision repair (NER) proteins (XPA and XPC). We found that the exposure to BaP or PCB126 alone could effectively increase the CYP1A1 activity and the XPA expression. BaP alone had a profound enhancement of MN formation. Compared with BaP alone, co-exposure to both BaP and PCB126 significantly enhanced the CYP1A1 activity and the formation of MN but reduced the expression of both XPA and XPC. The synergistic effect of PCB126 on BaP-induced MN formation was inhibited by alpha-naphthoflavone (ANF), an inhibitor of CYP1A1. Our findings suggest that PCB126 may enhance BaP-induced DNA damage and genotoxicity by increasing cytochrome P450 1A activity and decreasing the NER capacity.     

2,4-Diaminotoluene (2,4-DAT)-induced DNA damage, DNA repair and micronucleus formation in the human hepatoma cell line HepG2

2,4-Diaminotoluene (2,4-DAT) is a widely used industrial intermediate and human exposure is possible in the dye and plastics industries. We investigated the genotoxicity of the environmental pollutant, 2,4-DAT, in human HepG2 cells using the unscheduled DNA synthesis (UDS) test, the micronucleus (MN) assay and single-cell gel electrophoresis (SCGE). 2,4-DAT was first tested by the RNA synthesis inhibition test as a cytotoxicity assay: the IC(50) of 2,4-DAT was 5.2 mM after 20 h of exposure. The compound had a genotoxic effect at concentrations from 1.45 to 6.80 mM in both micronucleus and comet assays. In the micronucleus assay, the number of MN/1000 BNC was 3.5 times higher at a concentration of 6.80 mM 2,4-DAT than in the negative control. At the same concentration, DNA migration (SCGE) showed an Olive tail moment (OTM) of 3.56+/-0.45, as compared to 0.19+/-0.02 for the negative control. The UDS test detected genotoxic effects at lower concentrations than did the other assays (0.01-5 mM). The percentage of cells in repair increased in a concentration-dependent manner to a maximum of 57% at 1mM. At the highest concentration tested (5 mM), the NNG/cell score was 13.6+/-0.5 whereas it was -2.7+/-0.5 for the negative control. These data, based on various endpoints, show a midly genotoxic effect of 2,4-DAT in the HepG2 cells and confirm that this cell line is a suitable model to study the toxic effects of aromatic amines.     

3-Nitrobenzanthrone (3-NBA) induced micronucleus formation and DNA damage in human hepatoma (HepG2) cells

3-Nitrobenzanthrone (3-NBA), identified in diesel exhaust and in airborne particulate matter, is a potent mutagen in Salmonella, induces micronuclei formation in mice and in human cells and DNA adducts in rats. In the present study, we investigated the genotoxic potency of 3-NBA in human HepG2 cells using the micronucleus (MN) assay and the single cell gel electrophoresis (SCGE). 3-NBA caused a genotoxic effect at concentrations > or =12 nM in both assays. In the micronucleus assay, we found 98.7+/-10.3 MN/1000 BNC at a concentration of 100 nM 3-NBA in comparison to 27.3+/-0.6 MN/1000 BNC with the negative control. At the same concentration, the DNA-migration (SCGE) showed an Olive tail moment (OTM) of 2.7+/-0.45 and %DNA in the tail of 8.28+/-0.76; OTM and %DNA in the tail of cells treated with the negative control were 0.73+/-0.08 and 2.81+/-0.30, respectively. The results are discussed under consideration of former studies.     

Oxidative stress and cell-cycle change induced by coexposed PCB126 and benzo(a) pyrene to human hepatoma (HepG2) cells

Benzo(a)pyrene (BaP) never exists in the environment as a single compound but always coexists with other chemicals. These chemicals may affect the toxicity of BaP. Our previous study confirmed that polychlorinated biphenyls (PCBs), which were recently found coexisting with BaP in various environmental media, dramatically enhanced the genotoxicity of BaP. But the known mechanisms associated with this phenomenon are limited. Because BaP's genotoxicity is highly associated with its ability to induce the oxidative stress, we propose that the coexistence of PCBs may enhance BaP's genotoxicity by affecting BaP-induced oxidative stress. In this study, the HepG2 cells were treated with either BaP (50 μM), 3,3',4,4',5-pentachlorobiphenyl (PCB126) (0.01, 0.1, 1, and 10 nM), or pretreated with PCB126 followed by a coexposure to BaP and PCB126. We found that the exposure to BaP alone effectively increased the level of reactive oxygen species (ROS), glutathione (GSH), malondialdehyde (MDA), and the percentage of cells in G0/G1 phase, but decreased the percentage of S-phase cells. Compared to BaP alone, coexposure to both BaP and PCB126 effectively enhanced the levels of ROS and MDA as well as the percentage of cells in S phase, but decreased the levels of GSH and percentage of cells in G0/G1 phase. Our findings suggest that increasing oxidative stress and impairing the normal cell-cycle control may be mechanisms by which PCB126 enhances the genotoxity of BaP exposure.     

Genotoxic effects of ochratoxin A in human-derived hepatoma (HepG2) cells.

Ochratoxin A (OTA) is a widespread mycotoxin that occurs in many commodities from grains to coffee beans all over the world. Evidence is accumulating that OTA may cause cancer in humans. The compound was tested in micronucleus (MN) and single-cell gel electrophoresis (SCGE) assays in human-derived hepatoma (HepG2) cells and caused pronounced dose-dependent effects at exposure concentrations of 5 microg/ml and greater. On the contrary, no induction of His(+) revertants was found in Salmonella microsome assays with strains TA98 and TA100 with HepG2-derived enzyme (S9) mix in liquid incubation assays under identical exposure concentrations. Taken together, our results indicate that OTA is clastogenic in the human-derived cells. These findings support the assumption that this mycotoxin may cause genotoxic effects in hepatic tissue of humans.     

Flow cytometric evaluation of the contribution of ionic silver to genotoxic potential of nanosilver in human liver HepG2 and colon Caco2 cells

Exposure to nanosilver found in food‐ and cosmetics‐related consumer products is of public concern because of the lack of information about its safety. In this study, two widely used in vitro cell culture models, human liver HepG2 and colon Caco2 cells, and the flow cytometric micronucleus (FCMN) assay were evaluated as tools for rapid predictive screening of the potential genotoxicity of nanosilver. Recently, we reported the genotoxicity of 20 nm nanosilver using these systems. In the current study presented here, we tested the hypothesis that the nanoparticle size and cell types were critical determinants of its genotoxicity. To test this hypothesis, we used the FCMN assay to evaluate the genotoxic potential of 50 nm nanosilver of the same shape, composition, surface charge and obtained from the same commercial source using the same experimental conditions and in vitro models (HepG2 and Caco2) as previously tested for the 20 nm silver. Results of our study show that up to the concentrations tested in these cultured cell test systems, the smaller (20 nm) nanoparticle is genotoxic to both the cell types by inducing micronucleus (MN). However, the larger (50 nm) nanosilver induces MN only in HepG2 cells, but not in Caco2 cells. Also in this study, we evaluated the contribution of ionic silver to the genotoxic potential of nanosilver using silver acetate as the representative ionic silver. The MN frequencies in HepG2 and Caco2 cells exposed to the ionic silver in the concentration range tested are not statistically significant from the control values except at the top concentrations for both the cell types. Therefore, our results indicate that the ionic silver may not contribute to the MN‐forming ability of nanosilver in HepG2 and Caco2 cells. Also our results suggest that the HepG2 and Caco2 cell cultures and the FCMN assay are useful tools for rapid predictive screening of a genotoxic potential of food‐ and cosmetics‐related chemicals including nanosilver. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.     

Genotoxicity of acrylamide in human hepatoma G2 (HepG2) cells

The recent finding that acrylamide (AA), a carcinogen in animal experiments and a probable human carcinogen, is formed in foods during cooking raises human health concerns. The relevance of dietary exposure for humans is still under debate. The purpose of the study was to evaluate the possible genotoxicity of acrylamide in human hepatoma G2 (HepG2) cells, a cell line of great relevance to detect genotoxic/antigenotoxic substances, using single cell gel electrophoresis (SCGE) assay and micronucleus test (MNT). In order to clarify the underlying mechanism(s) we evaluated the intracellular generation of reactive oxygen species (ROS) and the level of oxidative DNA damage by immunocytochemical analysis of 8-hydroxydeoxyguanosine (8-OHdG). The involvement of glutathione (GSH) in the AA-induced oxidative stress was examined through treatment with buthionine sulfoximine (BSO) to deplete GSH. The results indicate that AA caused DNA strand breaks and increase in frequency of MN in HepG2 cells in a dose-dependent manner. The possible mechanism underlies the increased levels of ROS, depletion of GSH and increase of 8-OHdG formation in HepG2 cells treated with AA. We conclude that AA exerts genotoxic effects in HepG2 cells, probably through oxidative DNA damage induced by intracellular ROS and depletion of GSH.     

Spontaneous honeybee behaviour is altered by persistent organic pollutants

The effect of environmental pollutants on honeybee behaviour has focused mainly on currently used pesticides. However, honeybees are also exposed to persistent organic pollutants (POPs). The aim of this laboratory based study was to determine if exposure to sublethal field-relevant concentrations of POPs altered the spontaneous behaviour of foraging-age worker honeybees. Honeybees (Apis mellifera) were orally exposed to either a sublethal concentration of the polychlorinated biphenyl (PCB) mixture Aroclor 1254 (100?ng/ml), the organochlorine insecticide lindane (2.91?ng/ml) or vehicle (0.01% DMSO, 0.00015% ethanol in 1M sucrose) for 1–4 days. The frequency of single event behaviours and the time engaged in one of four behavioural states (walking, flying, upside down and stationary) were monitored for 15?min after 1, 2, 3 and 4 days exposure. Exposure to Aroclor 1254 but not lindane increased the frequency and time engaged in honeybee motor activity behaviours in comparison to vehicle. The Aroclor 1254—induced hyperactivity was evident after 1 day of exposure and persisted with repeated daily exposure. In contrast, 1 day of exposure to lindane elicited abdominal spasms and increased the frequency of grooming behaviours in comparison to vehicle exposure. After 4 days of exposure, abdominal spasms and increased grooming behaviours were also evident in honeybees exposed to Aroclor 1254. These data demonstrate that POPs can induce distinct behavioural patterns, indicating different toxicokinetic and toxicodynamic properties. The changes in spontaneous behaviour, particularly the PCB-induced chronic hyperactivity and the associated energy demands, may have implications for colony health.     

Perturbation by the PCB mixture aroclor 1254 of GABA(A) receptor-mediated calcium and chloride responses during maturation in vitro of rat neocortical cells

GABA(A) receptors are targets of highly chlorinated environmental chemicals and have important roles in developing neurons. As such, we examined effects of polychlorinated biphenyls (PCBs) on GABA(A) receptor responses in primary cultures of rat neocortical cells using fluorescence imaging techniques. Between days in vitro (DIV) 5 and 8, the effect of GABA(A) receptor stimulation switched from excitatory (Ca(2+) entry following a Cl(-) efflux; DIV /=7). GABA(A)-receptor-stimulated increases in [Ca(2+)](i) were diminished in a concentration-dependent (1-20 microM) manner following 1 h of exposure to the PCB mixture Aroclor 1254 (A1254), with significant reductions at concentrations as low as 2 microM. A1254 (1-20 microM) also led to concentration-dependent increases in basal [Ca(2+)](i), irrespective of DIV. A1254 (10 and 20 microM) significantly increased basal Ca(2+)(i); the Ca(2+)(i) was elevated to 426 +/- 39 nM by 20 microM A1254 but this concentration was not cytotoxic at 1 h. In addition, the mixture, A1254, as well as ortho- and non-ortho-chlorinated PCB congeners (IUPAC Nos. 4, 15, 126, and 138; 5-10 microM) individually decreased GABA(A)-stimulated Ca(2+)(i) responses and this tended not to depend on increases in basal Ca(2+)(i). In cultures DIV 7 and older, A1254 (20 microM) also impaired inhibitory GABA(A) responses as evidenced by an approximately 50% reduction of GABA(A)-stimulated Cl(-) influx (from approximately 6 to 8 mM net accumulation in controls). The results demonstrate that: (1) GABA(A) receptor increases in Ca(2+)(i) and Cl(-)(i) are inhibited by 2-20 microM A1254, regardless of whether the responses are at excitatory or inhibitory stages of development; (2) Ca(2+)(i) homeostasis in cortical cells is disrupted by 10 microM A1254; yet (3) disruption of excitatory GABA(A) responses by A1254 or PCB congeners does not necessarily depend on impaired Ca(2+) homeostasis. These novel observations suggest that GABA(A) receptor responses are a sensitive target for PCB effects in the rat developing nervous system.     

Effect of Sapthaparna (Alstonia scholaris Linn) in modulating the benzo(a)pyrene-induced forestomach carcinogenesis in mice

The chemopreventive effect of various doses of hydroalcoholic extract of Alstonia scholaris (ASE) was studied on the benzo(a)pyrene (BaP) induced forestomach carcinoma in female mice. The treatment of mice with different doses, i.e. 1, 2 and 4 mg/ml ASE in drinking water before, during and after the treatment with carcinogen, exhibited chemopreventive activity. The highest activity was observed for 4 mg/ml ASE, where the tumor incidence (93.33%) was reduced by 6.67%. Similarly, the tumor multiplicity reduced (61.29%) significantly (P<0.02) at 4 mg/ml in the pre-post-ASE treated group. However, the pre or post-treatment of mice with 4 mg/ml ASE did not show chemopreventive activity. These findings are corroborated by micronucleus assay, where treatment of mice with ASE before, during and after carcinogen treatment reduced the frequency of micronuclei (MN) in the splenocytes in a dose dependent manner. The MN frequency reached a nadir at 4 mg/ml ASE, the highest drug dose which showed maximum chemopreventive action. The ASE treatment not only reduced the frequency of splenocytes bearing one MN but also cells bearing multiple MN indicating the efficacy of ASE in inhibiting mutagenic changes induced by BaP. The pre or post-treatment of mice with 4 mg/ml ASE also significantly reduced the frequency of BaP-induced MN in the splenocytes of treated animals.     

DNA damage caused by extracts of chlorinated drinking water in human derived liver cells (HepG2)

Dong (D) lake and the Yangtze (Y) river are the main water supplies of the city of Wuhan, PR China. In the present study, the genotoxic effect of chlorinated drinking water (CDW) processed from raw water of D lake and Y river was evaluated in human HepG2 cells using the Comet assay and the micronucleus test. For that, HepG2 cells were exposed to XAD extracts of CDW corresponding to 0.167, 1.67, 16.7 and 167 ml CDW/ml cell culture. All CDW extracts caused a significant and dose-dependent increase of DNA migration in HepG2 cells. The level of DNA damage varied depending on the sampling time (season) and sampling site. The lowest concentration which caused a significant increase of DNA migration was 1.67 ml CDW/ml culture for water samples collected in August. Water samples collected in March showed their lowest observable effect levels in 167 ml and 16.7 ml CDW/ml culture for Y river and D lake, respectively. Additionally, significant increases of micronuclei (MN) frequencies were found in HepG2 cells after CDW treatment. However, in the MN assay the CDW samples collected in March exhibited higher genotoxicity than the August samples. In conclusion, HepG2 cells provide a useful tool for the detection of genotoxic effects of environmental mixtures.     

Absence of synergistic effects on micronucleus formation after exposure to electromagnetic fields and asbestos fibers in vitro.

Exposure of human amniotic fluid (AFC) cells to horizontally applied magnetic fields (hMF) of 50 Hz and 1 mT generated in a Helmholtz-coil system leads to a significant increase in micronucleus frequency (MN), without affecting cell proliferation. To investigate whether hMF-exposure has an additive or synergistic effect on the genotoxic capacity of asbestos fibers, MN induction was investigated in hMF pre-exposed cells, treated before or after with asbestos (1 microg/cm2). Neither synergistic nor additive effects on MN induction were observed. The results indicate, that under our experimental conditions, exposure to hMF and treatment with asbestos fibers possess genotoxic capability, but no interactive effects, in AFC cells.     

Investigation of the genetic toxicity by dextran-coated superparamagnetic iron oxide nanoparticles (SPION) in HepG2 cells using the comet assay and cytokinesis-block micronucleus assay

Dextran-coated superparamagnetic iron oxide nanoparticles (dextran-SPION) have been commercially used for medical applications, such as magnetic imaging or targeted drug delivery, etc. Despite of this usefulness, there are continuous controversies on safety/toxicity of various SPIONs. In the case of dextran-SPION, the reported adverse effects include the cytotoxicity, oxidative stress and DNA damage in many cell types. In this study, the genetic toxicity of dextran-SPION in human hepatoma (HepG2) cells was evaluated with comet assay and cytokinesis-block micronucleus (CBMN) assay. Dextran-SPION did not inhibit cell proliferation as a function of dose and time. The comet assay indicated that 5 μg/mL of dextran-SPION induced a significant DNA strand breaks. A dose-dependent increase of the micronucleus (MN) frequency was observed in CBMN assay. The formation of intracellular reactive oxygen species (ROS) in HepG2 cells was induced by dextran-SPION. The results demonstrated that dextran-SPION exerted genetic toxicity in HepG2 cells. Also, it suggests that a mechanism by which dextran-SPION triggers the DNA and chromosomal damage in HepG2 cells may be via the generation of intracellular ROS.     

DNA damage and metallothionein synthesis in human hepatoma cells (HepG2) exposed to cadmium.

Cadmium is an important heavy metal environmental toxicant, which is classified as a human carcinogen. The comet assay was used to evaluate the levels of DNA damage in a metabolically competent HepG2 cell line after treatment with low, non-cytotoxic and physiologically relevant concentrations of cadmium, alone and in combination with the dietary mutagen 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) and with the environmental mutagen benzo[a]pyrene (B(a)P). After exposure of the cells to 10, 100 and 1000 nM CdCl(2), a dose- and time-dependent increase of DNA damage was detected. Maximal damage was found after 12 h of treatment, but declined with further incubation with CdCl(2). The increased synthesis of metallothioneins on exposure to CdCl(2) up to 12 h suggests that they are responsible for the adaptation of HepG2 cells to the DNA damaging effects of CdCl(2). Co-treatment of the cells with CdCl(2) (10-1000 nM) and IQ (300 microM) induced a dose-dependent increase of DNA damage compared to cells treated with IQ alone. Co-genotoxic activity was also observed by increased formation of micronuclei in cells exposed to IQ and 1000 nM CdCl(2); at this concentration, CdCl(2) alone also induced micronuclei in HepG2 cells. Our results support the hypothesis that direct and indirect mechanisms are involved in cadmium-induced DNA damage.     

On the bioactivation and genotoxic action of fluoranthene

Fluoranthene (FA) was studied with respect to possible mechanisms of its high mutagenicity but low carcinogenicity, in comparison with the corresponding properties of benzo[a]pyrene (BaP), and with regard to the synergism of these two compounds shown by van Duuren and Goldschmidt (J Natl Cancer Inst 56, 1976, 1237). FA and BaP activated by S9 from Aroclor 1254 (PCB)-treated rats induce HPRT mutations in CHO cells with about equal effectiveness at the same exposure doses, which also lead to the same frequencies of repairable DNA adducts, enzyme-induced strand breaks being used as an indirect measure of adducts to DNA. FA was also shown to be an efficient inducer of SCE in human peripheral lymphocytes cocultivated with PCB-treated HepG2 cells or with liver cells from PCB-pretreated rats. For the induction of SCE, FA and BaP were shown to act additively. From metabolic studies with liver microsomes from C57B1/6 mice it is concluded that, whereas BaP induces the metabolism of BaP to the mutagenic epoxide, neither BaP nor FA is able to induce the metabolism of FA. In mutation experiments with V79 cells (XEM2) constitutive for P450 IA1 activity, BaP 7,8-diol but not FA 2,3-diol provokes a high frequency of HPRT mutations. In cells constitutive for P450 IA2 enzymatic activity FA and BaP are but weakly mutagenic and practically nonmutagenic, respectively. Due to the additivity of the genotoxic effects of FA and BaP, induction of an error-prone condition by the latter compound seems to be excluded. It therefore appears that for an explanation of the difference in carcinogenic potency between FA and BaP, indications that BaP but not FA acts as a promoter should be studied further.     

Adenosine triphosphatase activities in plasma liver membranes of rats treated with DDT and toxaphene

The effect of exposure to chlorinated insecticides (DDT and toxaphene) on Na+,K+-ATPase, Mg2+-ATPase and Ca2+-ATPase activities of the plasma membrane of hepatocytes was determined. Acute treatment with DDT (200 mg per kg body weight) or toxaphene (110 mg per kg body weight) produced a significant decrease in Na+,K+-ATPase activity (80% and 85%, respectively) 24 h after treatment. DDT also produced a 30% decrease in Mg2+-ATPase and Ca2+-ATPase activity, but toxaphene treatment did not modify these enzymes. The effect of exposure to daily doses of DDT (30 mg per kg body weight) or toxaphene (16.5 mg per kg body weight) for a period of 3.5 months was also studied. Animals were sacrificed at 15-day intervals and results showed that Na+,K+-ATPase activity decreased 80% from the beginning of each treatment and the activity remained low throughout the treatment period. DDT, but not toxaphene, also led to a decrease in Mg2+-ATPase (20%) and Ca2+-ATPase (35%) activity. The low values observed from the beginning remained low throughout the treatment period. We believe that the general mechanism of ATPase inhibition by organochloride compounds could be the result of its interaction with membrane lipid components, although some differences could arise from differences in their spatial structure.     

Comparison of four different treatment conditions of extended exposure in the in vitro micronucleus assay using TK6 lymphoblastoid cells

In the OECD Guideline 487, a total of four extended exposure treatment conditions are proposed for the in vitro micronucleus (MNvit) assay in the presence and absence of a cytokinesis block and with or without a recovery period. This guideline also states that rodent cell lines and human lymphocytes can be used as shown by many validated studies but that human cell lines such as TK6 and HepG2 are not yet validated. In this present study each extended exposure condition was characterized by investigation using TK6 cells and nine chemicals known to be able to induce micronucleus (MN) in rodent cell lines. The results revealed two concerns: six chemicals did not show significant MN induction in the ‘cytokinesis block without recovery period’; two aneugens showed no dose-dependent cytotoxicity in the ‘cytokinesis block with recovery period’. Further investigation revealed that 3–4 times higher spontaneous MN frequency than that in the other conditions is a possible reason for the low sensitivity, and this high spontaneous MN frequency was not observed in Chinese hamster lung cells under the identical treatment condition. With regard to the two conditions without cytokinesis block, two negative substances were evaluated and found to be negative, suggesting the validity of the TK6 test system for these conditions. Although our findings showed a few concerns for the treatment with cytokinesis block, the TK6 cells were considered to be a reliable cell line to be used for detecting potential inducers of MN in the in vitro micronucleus assay based on the overall results.