Micro-array chip analysis of carbonyl-metabolising enzymes in normal, immortalised and malignant human oral keratinocytes.

Enzymes involved in various protective and metabolic processes of carbonyl compounds were analysed utilising a micro-array method in a three-stage in vitro model for oral carcinogenesis involving cultured normal, immortalised and malignant human oral keratinocytes. A complete transcript profiling of identified carbonyl-metabolising enzymes belonging to the ADH, ALDH, SDR and AKR families is presented. Expression of 17 transcripts was detected in normal, 14 in immortalized and 19 in malignant keratinocytes of a total of 12,500 genes spotted on the micro-array chip. For the detected transcripts, about half were changed by cell transformation, and for the various enzyme families, differences in expression patterns were observed. The detected AKR transcripts displayed a conserved pattern of expression, indicating a requirement for the keratinocyte phenotype, while most of the detected SDRs displayed changed expression at the various stages of malignancy. The importance of multiple experiments in using a microarray technique for reliable results is underlined and, finally, the strength of the method in detecting co-expressed enzymes in metabolic pathways is exemplified by the detection of the formaldehyde-scavenging pathway enzymes and the polyol pathway enzymes.

Analysis of rat liver foci growth with a quantitative two-cell model after treatment with 2,4,5,3',4'-pentachlorobiphenyl.

A biologically based, quantitative foci-growth model was used to analyze the effect of 20 and 52 weeks treatment with 2,4,5,3', 4'-pentachlorobiphenyl (PCB118) on the development of enzyme-altered foci in rat liver initiated with partial hepatectomy and diethylnitrosamine. Hepatocyte proliferation was examined and the data were used in the selection of division rates for the computer simulations of foci growth. The bromodeoxyuridine-labeling index in foci was generally larger than in nonfocal tissue. A strong correlation was found between foci occurrence and proliferation in focal as well as nonfocal tissue, suggesting that cytotoxicity and regenerative proliferation are involved in the foci growth caused by PCB118. The foci growth model adequately simulated the foci data when certain assumptions were introduced. Given the general view that PCB118 is a nonmutagenic compound, the foci data could not be modeled assuming one homogeneous cell population, but was adequately fitted assuming two separate initiated cell populations that respond differently to the promotion stimulus. Other assumptions were a selective growth advantage for initiated cells during and shortly after the initiation treatment, and a transient increase of proliferation in focal hepatocytes at the first PCB administration in the higher dose groups. The model predicted an increased rate of focal cell death, at high doses, to adequately fit the foci data. These assumptions are supported by experimental data for other carcinogens, indicating the importance of studying cell kinetics in heterogeneous subpopulations of initiated cells in PCB-induced hepatocarcinogenesis.

Liver tumor-promoting activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in TCDD-sensitive and TCDD-resistant rat strains.

Risk assessment of dioxins is currently based on induction of liver tumors in rats. The toxicity of dioxins is characterized by large sensitivity differences among animal species and even strains of the same species, which complicates the risk assessment. The significance of these differences in dioxin-induced carcinogenicity is not known. We therefore studied the liver tumor-promoting activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the sensitive Long-Evans (L-E) and the resistant Han/Wistar (H/W) rats differing >1000-fold in their sensitivity to the acute lethaity of TCDD. Female rats were partially hepatectomized, initiated with nitrosodiethylamine, and treated with TCDD for 20 weeks. Altered hepatic foci (AHF) were stereologically quantitated using glutathione S-transferase P as a marker. AHF were significantly (P < 0.001) and dose dependently increased in L-E rats at 10 and 100 ng/kg/day, but in H/W rats only at 1000 ng/kg/day and above, indicating a remarkable (approximately 100-fold) sensitivity difference between L-E and H/W rats. The same sensitivity difference but 10-fold less foci were observed between nonhepatectomized/noninitiated L-E and H/W rats. Induction of AHF was related to hepatotoxicity but not to cytochrome P4501A1 activity in the liver. Liver TCDD concentrations were similar in both strains. H/W rats are exceptionally resistant to induction of AHF by TCDD, and the resistance is associated with an altered transactivation domain of the aryl hydrocarbon receptor. Genetic differences may account for significant interindividual/intraspecies sensitivity differences in dioxin-induced carcinogenesis. Understanding the role of transactivation domain of the aryl hydrocarbon receptor in carcinogenesis is therefore likely to improve dioxin risk assessment.

Induction of altered hepatic foci by a mixture of dioxin-like compounds with and without 2,2',4,4',5,5'-hexachlorobiphenyl in female Sprague-Dawley rats.

The hepatic tumor-promoting activity of a mixture of polyhalogenated aromatic hydrocarbons (PHAHs) was studied in a medium term two-stage initiation/promotion bioassay in female Sprague-Dawley rats. The PHAH mixture contained 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 1, 2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 2,3,4,7, 8-pentachlorodibenzofuran (PeCDF), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), 2,3',4,4',5-pentachlorobiphenyl (PCB 118), 2,3,3',4,4', 5-hexachlorobiphenyl (PCB 156), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) and covered >90% of the total toxic equivalents (TEQ) present in Baltic herring. To determine possible interactive effects of di-ortho-substituted PCBs, the PHAH mixture was tested with (PHAH+) and without (PHAH-) PCB 153. Rats were initiated by a diethylnitrosamine injection (30 mg/kg body wt i.p.) 24 h after a partial 23 hepatectomy. Six weeks after initiation, the PHAH mixtures were administered once a week by subcutaneous injections for 20 weeks. Treatment with the PHAH mixtures caused liver enlargement and an increased activity of the hepatic cytochrome P4501A1/2 and P4502B1/2. All PHAH exposure groups exhibited an increased occurrence of hepatic foci positive for the placental form of glutathione-S-transferase. In the PHAH-group dosed 1 microgram TEQ/kg body wt/week, the volume fraction of the liver occupied by foci was significantly lower compared to the TEQ equivalent dosed TCDD group (3.8 vs 8.7%). The volume fraction was significantly increased in the groups treated with 0.5, 1, or 2 micrograms TEQ/kg body wt/week of the PHAH+ mixture (4.5, 5.2, and 6.6%, respectively) compared to the corn oil group (2.0%), but to a lower extent than expected on basis of the TEQ doses. Overall, the TEQ-based administered dose overestimated the observed tumor-promoting effects of this PHAH mixture. The applicability of the toxic equivalency factor concept, the role of differences in toxicokinetic properties and interactive effects of PCB 153 on hepatic deposition of the dioxin-like congeners are discussed.

Interactive effects of three structurally different polychlorinated biphenyls in a rat liver tumor promotion bioassay.

Interactive effects between the non-ortho-substituted 3,3', 4,4', 5-pentachlorobiphenyl (PCB126), the mono-ortho-substituted 2,3,3',4, 4'-pentachlorobiphenyl (PCB105), and the di-ortho-substituted 2,2',4, 4',5,5'-hexachlorobiphenyl (PCB153) were studied in an initiation/promotion bioassay. Female Sprague-Dawley rats were injected with 30 mg/kg ip of N-nitrosodiethylamine 24 h after partial hepatectomy. Five weeks later, weekly sc administrations of the three PCBs in 15 systematically selected dose combinations started. After 20 weeks of administration, the animals were killed and the livers were analyzed for areas expressing placental glutathione-S-transferase as a marker of preneoplastic foci. In addition, concentration of liver and kidney retinoids and plasma retinol was analyzed, as well as body and organ weights, plasma transaminases, and induction of hepatic cytochrome P450 1A1/2 (CYP1A1/2) and CYP2B1/2 activities. Data were analyzed with a multivariate method. At the doses applied in this study, weak antagonism was observed between PCB126 and PCB153 for effects on volume fraction of foci, number of foci/cm3, concentration of plasma retinol and liver retinoids, relative liver weight, and induction of CYP2B1/2 activity. Weak antagonism was also observed between PCB126 and PCB105 for effects on volume fraction of foci, number of foci/cm3, and plasma retinol concentration. No interactions other than pure additivity were observed between PCB105 and PCB153. Synergism was not observed within the dose ranges investigated in this study. Knowledge of interactive effects is important for risk assessment of environmental mixtures of dioxin-like compounds. Antagonism between congeners generally results in risk assessments that overestimate human risk. The significance to human risk assessment of the relatively weak antagonism observed in this study is however unclear, considering many other uncertainties involved in the toxic equivalency factor (TEF) concept. A change of the TEF concept for risk assessments of dioxin-like substances is not motivated based on the results of this study.

Initiation and promotion of altered hepatic foci in female rats and inhibition of cell-cell communication by the imidazole fungicide prochloraz.

The imidazole fungicide prochloraz (1-[N-propyl-N-2-(2,4,6-trichlorophenoxy) ethyl carbamoyl] imidazole) was investigated for its ability to inhibit gap junctional intercellular communication in the scrape-loading/dye-transfer assay in IAR 20 rat liver epithelial cells and for effects on the initiation and promotion stages of hepatocarcinogenesis. Female Sprague-Dawley rats initiated with N-nitrosodiethylamine 24-hr after partial hepatectomy were administered prochloraz five days a week by oral gavage (30 or 150 mg/kg) for 10 weeks. Altered hepatic foci (AHF) were analyzed by quantitative stereology from liver sections stained for gamma-glutamyltranspeptidase (GGT) and glutathione S-transferase P (GST-P). The fungicide was also studied for its ability to initiate the development of GGT-positive AHF in rat liver. The in vitro studies showed prochloraz to be an inhibitor of cell-cell communication in the test system used. In the in vivo studies, prochloraz showed no effect on the initiation of GGT-positive foci. However, significant increases in the percentage of liver tissue occupied by GGT-positive AHF and the number of GST-P-positive AHF per cm3 in initiated animals were recorded in the low dose group. The present data suggest that prochloraz acts as a weak tumour promoter of hepatocarcinogenesis but does not initiate this process.

Inhibition of cell-cell communication by methylsulfonyl metabolites of polychlorinated biphenyl congeners in rat liver epithelial IAR 20 cells.

The effects of three polychlorinated biphenyl (PCB) congeners and their six methylsulfonyl (MeSO2)-metabolites on cell communication have been investigated in the scrape-loading/dye-transfer assay in IAR 20 rat liver epithelial cells. The results demonstrated that at non-cytotoxic concentrations 2,2',4',5-tetrachlorobiphenyl, 2,2',4',5,5'-pentachlorobiphenyl (2,2',4',5,5'-pentaCB), 2,2',4',5,5',6-hexachlorobiphenyl (2,2',4',5,5', 6-hexaCB), and their 3- and 4-MeSO2 derivatives completely inhibited the cell communication within 1 h. 4-MeSO2-2,2',4',5,5'-pentaCB and 4-MeSO2-2,2',4',5, 5',6-hexaCB appeared to inhibit the cell communication at slightly lower concentration than their parental PCB congeners and 3-MeSO2 derivatives. The results show that 3- and 4-MeSO2 derivatives of the PCB congeners tested inhibit gap junction intercellular communication at about the same potency as their parental compounds. Since inhibition of cell communication is often observed after treatment with many tumor promoters, our findings suggest that the metabolites may also act as tumor promoters.

Session 4: Application of Molecular Biology inIn Vitro Toxicology.

The implementation of molecular biology in vitro toxicology has been a challenge over the last years. The present session had the special task of discussing how this new technique could be integrated and used by toxicologists. The session consisted of five different lectures. The first lecture was an overview concerning the role of cytokines in toxicology. This lecture was followed by a discussion on the topic of how effects of xenobiotics on target cells reflect the toxicity in vivo. In the third lecture the use of gene expression pattern as molecular endpoint for mechanistic toxicity was discussed, and it was suggested that array of systems would be a valuable tool in the near future. This lecture was followed by two mechanistically related lectures dealing with lead-induced alteration of gene expression in the retina, followed by a discussion concerning mechanisms of signal transduction mediated by the dioxin receptor. These lectures demonstrated that there is a need to understand the complexity before we simplify a biological process. In summary, the session discussed future possibilities for studying toxic responses in a more mechanistically direct way and how pattern recognition of altered gene expression could be an important tool in predicting toxic phenotypic alterations.

Promotion of enzyme altered foci in female rat 2,3,3',4,4',5-hexachlorobiphenyl.

The tumor promoting activity of 2,3,3',4,4',5-hexachlorobiphenyl (PCB 156) was studied in an initiation/promotion bioassay in female Sprague-Dawley rats initiated with N-nitrosodiethylamine after partial hepatectomy. PCB 156 (50, 300, 1500, or 7500 microg/kg body weight/week) was administered by once-weekly subcutaneous injections for 20 weeks. Some high dose animals were left without treatment for an additional 20 weeks to study posttreatment effects. The volume fraction of the liver occupied by glutathione S-transferase P-positive foci was significantly increased to 2.9, 3.3, and 12% at 300, 1500, and 7500 microg/kg body weight/week, respectively, compared to 1.2% in the controls. The volume fraction was 43% in the high dose group 20 weeks after treatment was stopped, probably reflecting the slow body clearance of PCB 156 as indicated by the sustained liver and adipose tissue concentrations. Treatment with PCB 156 following initiation caused decreased body weight gain, thymic atrophy, liver enlargement, induction of hepatic cytochrome P450 1A1/2 (CYP1A1/2) and CYP2B1/2 activities, histopathological effects, and increased activities of aspartate aminotransferase and gamma-glutamyltransferase in plasma. These results show that PCB 156 can enhance the growth of altered foci in rat liver and probably act as a tumor promoter of hepatocarcinogenesis. Based on promotional activity a relative potency of PCB 156 to 2,3,7, 8-tetrachlorodibenzo-p-dioxin of 0.0001-0.001 is proposed.

The ability to alter the gap junction protein expression outside GST-P positive foci in liver of rats was associated to the tumour promotion potency of different polychlorinated biphenyls.

The results demonstrate different modes of action by a dioxin-like polychlorinated biphenyl (PCB 126) and a non dioxin-like PCB (PCB 153) in the alteration of connexin (cx) 26 and cx 32 expression outside GST-P positive foci in liver of female Sprague-Dawley rats, treated according to an initiation-promotion protocol. A decreased relative amount of immunopositive cx 26 and cx 32 spots in the parenchymal cell plasma membranes was observed after treatment with the potent tumour promoters PCB 126 or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). No reduction of cx 26 or cx 32 was noted after administration with the weaker tumour promoters PCB 153 or PCB 118 (PCB 118; both dioxin- and non dioxin-like). Additionally, we found that the down-regulation of connexins also occurred in rats treated with PCB 126 or TCDD without partial hepatectomy and initiation with nitrosodiethylamine. In summary, the results indicate that the ability to reduce the gap junction protein level in liver of rats can be associated to the tumour promotive potency of the different PCB-congeners and TCDD.

Promotion of altered hepatic foci by 2,3',4,4',5-pentachlorobiphenyl in Sprague-Dawley female rats.

The tumor promotion potential of 2,3',4,4',5-pentachlorobiphenyl (PCB-118) was studied in a two-stage initiation/promotion bioassay in female Sprague-Dawley rats. The animals were initiated by intraperitoneal administration of N-nitrosodiethylamine after partial hepatectomy. After 5 weeks of recovery, the promotion period commenced by once-weekly subcutaneous administrations of PCB-118 at six dose levels (10, 40, 160, 640, 2500, and 10,000 microg/kg body weight/week) for 20 weeks. In addition, three of these dose levels (40, 640, and 10,000 microg/kg body weight/week) were administered for 52 weeks. Evaluation of hepatic foci positive for glutathione S-transferase P demonstrated that the mono-ortho chlorine substituted congener PCB-118 significantly increased the number of foci/cm3 of liver in the two highest dose groups after 20 weeks, but did not significantly increase the percentage of the liver occupied by foci. After 52 weeks of treatment, both the percentage and the number of foci/cm3 were significantly increased in the highest dose group. A toxic equivalency factor based on foci development during 20 weeks of treatment would be less than 0.00002. Altered relative liver and thymus weights were observed after treatment with both substances as well as an induction of methyl cholanthrene- and phenobarbital-inducible isoenzymes of cytochrome P450 monooxygenase. These results show that PCB-118 has a potency to enhance foci growth in rat liver, although the potency is low compared to that of structurally related compounds.

Altered function, localization and phosphorylation of gap junctions in rat liver epithelial, IAR 20, cells after treatment with PCBs or TCDD.

Three different PCB-congeners 3,4,5,3',4'-pentachlorobiphenyl (IUPAC no. 126), 2,4,5,2',4',5'-hexachlorobiphenyl (IUPAC no. 153) and 2,4,5,3',4'-pentachlorobiphenyl (IUPAC no. 118) were investigated for possible structure-activity relationships in altering gap junction intercellular proteins. All tested PCB-congeners and TCDD decreased the gap junctional intercellular communication in IAR 20 cells, but at different treatment periods, suggesting different modes of action. The presence of the Cx43-P(2) band, a phosphorylated isoform of Cx43, was associated with a functional communication. A reduced Cx43 mRNA level was noted after 48 h of exposure with PCB 126, PCB 118 and TCDD. In summary, the non dioxin-like PCB 153 can decrease gap junctional intercellular communication rapidly by reducing the phosphorylated isoform of Cx43, whereas the dioxin-like PCB 126 and TCDD reduce the communication slowly by decreasing the mRNA level of Cx43, resulting in a reduced Cx43 protein level (which includes the P(2)-band). The mixed inducing PCB-congener, PCB 118, can act both as the dioxin-like and the non dioxin-like PCBs in gap junction regulation.

Mechanistical studies of the inhibition of intercellular communication by organochlorine compounds.

Many hydrocarbons are environmental pollutants that, due to their lipophilicity and chemical stability, accumulate in biological systems including milk and body fat. A number of investigations have demonstrated that many organochlorine compounds can act as tumour promoters in vivo and inhibit gap junctional intercellular communication between cells in culture. In the present study we have investigated the dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), different polychlorinated biphenyls, chlorinated paraffins and the pesticide endosulfan. Using techniques of scrape loading dye/transfer and Western blot analysis the function, expression and phosphorylation of different connexins in vitro and in vivo were studied. The results show a good correlation between the ability to act as a tumour promoter and to interfere with gap junctional intercellular communication. All tested compounds inhibited the intercellular communication in a liver derived cell line (IAR 20). However, the results show that the time to inhibition varies between the different agents. Endosulfan and chlorinated paraffins inhibit the communication within one hour, whereas dioxin like substances need to expose the cells for 48 hours before the communication is affected.

Interaction of 3,4,5,3',4'-pentachlorobiphenyl and 2,4,5,2',4',5'-hexachlorobiphenyl in promotion of altered hepatic foci in rats.

This study was undertaken to investigate tumour promoting interactions of 2,4,5,2',4',5'-hexachlorobiphenyl (PCB 153) and 3,4,5,3',4'-pentachlorobiphenyl (PCB 126) in female Sprague-Dawley rats. Five weeks before the promotion treatment, the rats were partially hepatectomized and initiated with nitrosodiethylamine. The test substances were administered by weekly, subcutaneous injections for 20 weeks. The results from this study suggest that treatment with a combination of these two congeners causes a more than additive effect on the formation of gamma-glutamyltranspeptidase-positive hepatic foci. Co-exposure to PCB 126 and PCB 153 caused a dose-dependent reduction of the PCB 153-induced CYP2B1/B2-activity in these livers.

Liver tumour promoting activity of 3,4,5,3',4'-pentachlorobiphenyl and its interaction with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

This study was undertaken to compare the tumour promoting effects induced by 3,4,5,3',4'-pentachlorobiphenyl (PCB 126) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In addition, interactive effects in rats treated with combinations of PCB 126 and TCDD were studied. Partially hepatectomized female Sprague-Dawley rats were initiated with nitrosodiethylamin. After 5 weeks of recovery the promotion treatment started and continued for 20 weeks. The results from the present study demonstrate that PCB 126 elicit approximately 10% of TCDD's tumour promoting activity measured as enhancement of the development of gamma-glutamyl-transpeptidase-positive altered hepatic foci in the liver. The factor required for the PCB to match the response of TCDD was adopted as a toxic equivalency factor and was in this case 0.1, which is the same as the factor suggested by Ahlborg et al. (1994). In the groups treated with a mixture of PCB 126 and TCDD the tumour promoting effect indicated an additive response. This result suggests that PCB 126 and TCDD act by the same mechanistical pathway, which in turn, supports that the toxic equivalency factor-concept can be used for TCDD-like tumour promoters.

Alteration in expression of gap junction proteins in rat liver after treatment with the tumour promoter 3,4,5,3',4'-pentachlorobiphenyl.

Polychlorinated biphenyls (PCBs) are industrial chemicals which are highly persistent and widely distributed in the environment. We have previously shown that 3,4,5,3',4'-pentachlorobiphenyl (PCB 126) is a potent tumour promoter in two separate 20 week initiation-promotion studies. In the present study, rat livers from these two studies were further investigated for connexin expression. The results demonstrated that treatment with PCB 126 caused a decrease in the amount of the two major liver connexins, cx 26 and cx 32, in livers of treated animals. This reduction was also prominent after treatment at low doses, although gamma-glutamyl transpeptidase-positive foci had not developed in these livers. The quantity of cx 26 and cx 32 in immunostained liver sections was determined using a computerized fluorescence image analyzer. Western blot analysis of liver extracts confirmed these results. No changes in the RNA levels in the treated rats were seen, suggesting that the down-regulation of cx 26 and cx 32 is post-transcriptional.

Two inhibitors of gap junctional intercellular communication, TPA and endosulfan: different effects on phosphorylation of connexin 43 in the rat liver epithelial cell line, IAR 20.

The skin tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the chlorinated insecticide, endosulfan, are two potent inhibitors of gap junctional intercellular communication. In the present study the effects of TPA and endosulfan on cell communication have been investigated in IAR 20 rat liver epithelial cells, as well as the effects of these compounds on connexin 43 (cx43), the main gap junction protein in this cell line. The results clearly demonstrate that at non-toxic doses both compounds inhibited the cell communication by at least 90% within 5 min. The communication was partially restored after 4 h of TPA exposure and almost fully restored by 24 h, whereas in endosulfan-exposed cells the communication was completely down-regulated for the whole exposure-period of 24 h. Immunoblots of IAR 20 cell extracts indicated that TPA initially caused an increased phosphorylation of cx43. A normal phosphorylation pattern was observed after 4 h when the cell communication was restored. Immunoblot analysis after endosulfan-exposure showed a slightly increased phosphorylation of cx43 after 10 min treatment, gradually followed by dephosphorylation during the rest of the 24 h treatment period. Immunostaining of IAR 20 cells showed that both compounds caused a rapid disappearance of cx43 from the cell membrane. After 4 h of exposure immunofluorescent cx43-plaques started to reappear in the cell membrane, although less pronounced in endosulfan-treated cells. However, after 24 h of endosulfan-exposure a high number of cx43-spots was demonstrated. These results indicate that different mechanisms are responsible for the inhibition of gap junctional intercellular communication induced by TPA and by endosulfan, at least during the later part of the 24 h exposure-period. TPA causes a marked hyperphosphorylation of cx43, whereas endosulfan increases phosphorylation initially only slightly but longer exposure-periods lead to hypophosphorylation. Thus, phosphorylation as well as dephosphorylation seem to be important factors involved in the regulation of the function of cx43 in this cell line.

Enhancement of altered hepatic foci in rat liver and inhibition of intercellular communication in vitro by the pyrethroid insecticides fenvalerate, flucythrinate and cypermethrin.

Male Sprague-Dawley rats dosed with N-nitrosodiethylamine (NDEA) 24 h after two-thirds partial hepatectomy were treated with the pyrethroid insecticides fenvalerate, flucythrinate or cypermethrin in the diet for 20 weeks. Altered hepatic foci were analyzed by quantitative stereology from paraffin-embedded sections stained for gamma-glutamyltranspeptidase (GGT) or glutathione S-transferase P (GST-P). The present results demonstrate that the pyrethroids tested all enhance the development of NDEA-initiated, GGT-positive foci in rat liver at non-hepatotoxic doses. On the contrary, the volume fractions of GST-P-positive foci were not elevated as compared to the control group. The three pyrethroids tested all inhibited the transfer of Lucifer Yellow CH between WB-F344 rat liver epithelial cells in culture, supporting the increase of GGT-positive foci and suggesting that these substances can act as tumour promoters. The discrepancy between the results from analyses using GGT or GST-P as markers emphasizes the importance of understanding the mechanism underlying the expression of different markers for preneoplastic lesions and the importance of such effects in tumour promotion.