Setting the stage for next-generation risk assessment with non-animal approaches: the EU-ToxRisk project experience.

In 2016, the European Commission launched the EU-ToxRisk research project to develop and promote animal-free approaches in toxicology. The 36 partners of this consortium used in vitro and in silico methods in the context of case studies (CSs). These CSs included both compounds with a highly defined target (e.g. mitochondrial respiratory chain inhibitors) as well as compounds with poorly defined molecular initiation events (e.g. short-chain branched carboxylic acids). The initial project focus was on developing a science-based strategy for read-across (RAx) as an animal-free approach in chemical risk assessment. Moreover, seamless incorporation of new approach method (NAM) data into this process (= NAM-enhanced RAx) was explored. Here, the EU-ToxRisk consortium has collated its scientific and regulatory learnings from this particular project objective. For all CSs, a mechanistic hypothesis (in the form of an adverse outcome pathway) guided the safety evaluation. ADME data were generated from NAMs and used for comprehensive physiological-based kinetic modelling. Quality assurance and data management were optimized in parallel. Scientific and Regulatory Advisory Boards played a vital role in assessing the practical applicability of the new approaches. In a next step, external stakeholders evaluated the usefulness of NAMs in the context of RAx CSs for regulatory acceptance. For instance, the CSs were included in the OECD CS portfolio for the Integrated Approach to Testing and Assessment project. Feedback from regulators and other stakeholders was collected at several stages. Future chemical safety science projects can draw from this experience to implement systems toxicology-guided, animal-free next-generation risk assessment.

Hazard assessment of nitrosamine and nitramine by-products of amine-based CCS: alternative approaches.

Carbon capture and storage (CCS) technologies are considered vital and economic elements for achieving global CO2 reduction targets, and is currently introduced worldwide (for more information on CCS, consult for example the websites of the International Energy Agency (http://www.iea.org/topics/ccs/) and the Global CCS Institute (http://www.globalccsinstitute.com/)). One prominent CCS technology, the amine-based post-combustion process, may generate nitrosamines and their related nitramines as by-products, the former well known for their potential mutagenic and carcinogenic properties. In order to efficiently assess the carcinogenic potency of any of these by-products this paper reviews and discusses novel prediction approaches consuming less time, money and animals than the traditionally applied 2-year rodent assay. For this, available animal carcinogenicity studies with N-nitroso compounds and nitramines have been used to derive carcinogenic potency values, that were subsequently used to assess the predictive performance of alternative prediction approaches for these chemicals. Promising cancer prediction models are the QSARs developed by the Helguera group, in vitro transformation assays, and the in vivo initiation-promotion, and transgenic animal assays. All these models, however, have not been adequately explored for this purpose, as the number of N-nitroso compounds investigated is yet too limited, and therefore further testing with relevant N-nitroso compounds is needed.

WITHDRAWN: Hazard assessment of nitrosamine and nitramine by-products of amine-based CCS: an alternative approach.

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Evaluation of an alternative in vitro test battery for detecting reproductive toxicants.

The application of alternative methods in developmental and reproductive toxicology is challenging in view of the complexity of mechanisms involved. A battery of complementary test systems may provide a better prediction of developmental and reproductive toxicity than single assays. We tested twelve compounds with varying mechanisms of toxic action in an assay battery including 24 CALUX transcriptional activation assays, mouse cardiac embryonic stem cell test, ReProGlo assay, zebrafish embryotoxicity assay, and two CYP17 and two CYP19 activity assays. The battery correctly detected 11/12 compounds tested, with one false negative occurring, which could be explained by the absence of the specific mechanism of action of this compound in the battery. Toxicokinetic modeling revealed that toxic concentrations were in the range expected from in vivo reproductive toxicity data. This study illustrates added value of combining assays that contain complementary biological processes and mechanisms, increasing predictive value of the battery over individual assays.

Carcinogenic activity of benzo[a]pyrene in a 2 year oral study in Wistar rats.

Because of the relatively high human oral exposure to polycyclic aromatic hydrocarbons (PAHs) compared to the inhalation exposure, the known carcinogenicity of this type of compounds and the limited data from oral studies available with polycyclic aromatic hydrocarbons, an oral carcinogenicity study was performed using benzo[a]pyrene (B[a]P) as a PAH representative. Wistar rats, 52 animals per sex and group were exposed daily (5 days a week) to 0, 3, 10 or 30 mg B[a]P/kg bw/day by gavage for 104 weeks and were subject to gross- and histopathology. The main tumours observed were hepatocellular carcinomas and forestomach tumours. Other tumours induced in this study were tumours of the auditory canal, skin and appendages, oral cavity, small intestine, kidney, and soft tissue sarcomas. For hepatocellular carcinomas and forestomach tumours, the BMDL10 were 3 and 1 mg/kg bw/day, respectively. The incidence of altered hepatic foci was increased in the 3mg/kg bw/day group. The increase in liver tumours is considered the most relevant effect for human risk assessment in terms of pathogenesis and sensitivity, and is proposed as the basis for human cancer risk assessment for oral PAH exposure.

Evaluation of inhalation TTC values with the database RepDose.

The thresholds of toxicological concern (TTCs) define limit values for substances of unknown toxicity below which dietary intake is considered to be of no concern to human health. The TTC concept has already been used for risk assessment of e.g. food contaminants or flavoring substances and is in discussion to be applied to other classes of compounds such as cosmetic ingredients, household products, non-relevant metabolites in drinking water, and impurities in pharmaceuticals. The present publication aimed to evaluate whether the current TTC concept can also be applied to define limit values for inhalation exposure, using a data set of 203 industrial chemicals from the database RepDose. It has been shown, that the NOEC values in classes 1, 2, and 3 are distributed over six orders of magnitude resulting in a considerable overlap between the distribution curves for the three classes. Inhalation thresholds for Cramer classes 1 (compounds likely to be of low-toxicity), 2 (compounds likely to be of moderate toxicity), and 3 (compounds suspect for high toxicity) were analyzed close to the approach described by Munro for oral TTCs. The 5th percentiles NOEC of Cramer classes 1-3 result in thresholds of 1.5×10(-3) ppm for Cramer class 1 and 2.2×10(-5) ppm for Cramer class 3. A threshold could not be derived for class 2 because of the small number of compounds available. If calculated as body doses, the inhalation thresholds for classes 1 and 3 (71 and 4 µg/person/d, respectively) are considerably lower than the oral thresholds derived by Munro (1800 and 90 µg/person/d). It has been shown that one reason for this difference is the high sensitivity of the respiratory tract to local effects. In a next step, the values obtained were further refined. If organophosphates or compounds with structural alerts for genotoxicity are excluded, the TTC in Cramer class 1 increases, whereas the TTC in Cramer class 3 remains the same. Based on these analyses two inhalation TTCs for non-genotoxic compounds are proposed: 3.6×10(-3) ppm (180 µg/person/d) for Cramer class 1 and 2.4×10(-5)ppm (4 µg/person/d) for Cramer class 3.

Use of read-across and tiered exposure assessment in risk assessment under REACH--a case study on a phase-in substance.

REACH requests the exploration of alternative strategies for hazard identification before resorting to (in vivo) testing. Here, we combined read-across as non-testing strategy with a tiered exposure assessment for the risk characterisation of 1-methoxypropan-2-ol (PGME) as a representative for phase-in substances to be registered under REACH. Read-across from the selected source substances provided data which were comparable with experimental data available for target substance PGME, resulting in a realistic starting point for both qualitative and quantitative risk assessment. Greater variability was observed in the exposure estimates from a first Tier model (ECETOC TRA) or less conservative further Tier models (Stoffenmanager; RISKOFDERM), when these results were compared with results from a data-rich approach using measured data. When safe use of chemicals cannot be demonstrated with these approaches, refinement can be introduced in the estimation of hazard and exposure, or both. In view of the variability associated with exposure modeling, it may often add more value to invest in realistic exposure data than in toxicity studies, apart from animal welfare considerations.

REACH, non-testing approaches and the urgent need for a change in mind set.

The objectives of REACH cannot be achieved under the current risk assessment approach. A change in mind set among all the relevant stakeholders is needed: risk assessment should move away from a labor-intensive and animal-consuming approach to intelligent and pragmatic testing, by combining exposure and hazard data effectively and trying to group chemicals (category approaches). The focus should be on reducing the overall uncertainties of 30,000 chemicals while acknowledging the existence of the uncertainty paradox: reducing uncertainty in the assessment of individual chemicals following the classical chemical-by-chemical approach as we have in previous decades will result in a prolongation of uncertainty for the entire group of 30,000 chemicals as a whole. With the first REACH registration deadline (2010) rapidly approaching, a mind set change is urgently needed. We can speed up the regulatory acceptance process, starting with the maximum use of currently available exposure and hazard data, tools and models. Optimal use should also be made of experimental exposure and hazard data generated under REACH. Only such an approach will make it possible to obtain a sufficient level of information within the time frame of REACH. A much more intensive dialogue between stakeholders is necessary.

A simple method for quantitative risk assessment of non-threshold carcinogens based on the dose descriptor T25.

This report provides guidance for using the dose-descriptor T25 from animal studies as a basis for quantitative risk characterisation of non-threshold carcinogens. T25 is presently used within the European Union for setting specific concentration limits for carcinogens in relation to labelling of preparations (formulations). The T25 is defined as the chronic dose rate which will give 25% of the animals tumours at a specific tissue site, after correction for spontaneous incidence, within the standard life-time of that species. The T25 is converted to the corresponding human dose descriptor, HT25, by dividing it with the appropriate scaling factor for interspecies dose scaling based on comparative metabolic rates. Subsequently, the human dose (expressed in mg per kg body-weight per day) is calculated from the available exposure data. The corresponding human life-time cancer risk is then obtained by using linear extrapolation by dividing the exposure dose with the coefficient (HT25/0.25). The results with this new method, which can easily be calculated without computer programmes, are in excellent agreement with results from computer-based extrapolation methods such as the linearised multistage model and the benchmark method using LED10, even though the present method only takes into consideration one single dose-response point. To overcome possible shortcomings of the present method, the estimated life-time risks are proposed to be accompanied by a commentary statement giving an overall evaluation of data that may have bearing on the carcinogenic risk and that may indicate whether the real human risk is likely to be higher or lower than the calculated life-time risk. By using the present guidance and a harmonized set of criteria and default values, the calculation of life-time cancer risk should be transparent and easy to comprehend.

Detection of immunotoxicity of benzo[a]pyrene in a subacute toxicity study after oral exposure in rats.

In an extended OECD 407 study protocol, including immune parameters, male Riv:Tox Wistar SPF rats were treated for 35 days with benzo[a]pyrene (B[a]p) (3, 10, 30, or 90 mg/kg body weight) by gavage. Oral administration of B[a]p in rats resulted not only in general toxicity, as indicated by the effects on body weight, but also in immunotoxicity, as indicated by the effects on bone marrow, thymus, spleen, and lymph nodes. Oral B[a]p induced a dose-related decrease in thymus weight (at 10, 30, and 90-mg/kg). Lymph node weights (popliteal, mandibular, and mesenteric) were decreased in the 90-mg/kg rats only. Histologically, indications for cortical atrophy were noted in the thymuses of the 30- and 90-mg/kg dose groups, which was confirmed by morphometric analysis. Nucleated spleen and bone marrow cell counts were decreased in the 90-mg/kg group. Both the absolute number (90 mg/kg) and relative number (10, 30, and 90 mg/kg) of B cells in the spleen were decreased. Red blood cell (RBC) and white blood cell (WBC) counts were significantly decreased; for the WBC at 90 mg/kg, and for the RBC at 10, 30, and 90 mg/kg. The absolute number of lymphocytes and eosinophilic granulocytes was decreased in the 90-mg/kg group, while the absolute number of monocytes was increased in the 10- and 30-mg/kg dose groups. Serum immunoglobulin levels showed a decrease of IgM and IgA after treatment of the animals with 30 and 90 mg/kg, respectively. The highest dose of B[a]p treatment (90 mg/kg) resulted in a significant decrease of natural killer (NK)-cell activity in the spleen. Most toxic effects were only observed in the highest-dose group (90 mg/kg), but compared to the general toxicity, some parameters indicating immunotoxic effects were also affected at lower doses (10 and 30 mg/kg). In conclusion, immunotoxicity of B[a]p can be detected using parameters of the immune system such as described in the recently updated OECD 407 guideline. In the present study thymus weight changed and spleen B-cell populations were affected at a dose of 10 mg/kg, a level where no overt general toxicity was noted.

Evaluation of the Emu-pim-1 transgenic mouse model for short-term carcinogenicity testing.

The value of the chronic rodent carcinogenicity assay in adequately predicting cancer risk in humans has become a matter of debate over the past few years. Therefore, more rapid and accurate alternative tests are urgently needed. Transgenic mouse models, those harboring genetic changes that are relevant to the multistage cancer process, may provide such alternative tests. Transgenic Emu-pim-1 mice, developed by Berns and coworkers in 1989, contain the pimn-1 oncogene, which is expressed at elevated levels in their lymphoid compartments. As a result, these mice are predisposed to the development of T-cell lymphomas. Because of the low incidence of spontaneous tumors and the increased sensitivity to N-ethyl-N-nitrosourea-induced carcinogenesis, Emu-pim-1 mice were suggested to be one of the first potential and attractive candidates to be used in short-term carcinogenicity testing. In the present article, the results from 2 recent short-term assays (with mitomycin C and x-rays) are briefly presented, together with a review of all 11 performed bioassays and their corresponding histopathologic and molecular data. The overall results allow the first evaluation of the Emu-pim-1 mouse model with regard to its usefulness in short-term carcinogenicity testing. It has been shown that the model is primarily suitable as a sensitive short-term assay for genotoxic carcinogens that not only induce (at least) gene mutations and/or large deletions and rearrangements but that also sufficiently target the lymphoid system. However, the Emu-pim-1 mice lack sufficient sensitivity to justify their routine use in short-term carcinogenicity testing in general.

Use of E mu-PIM-1 transgenic mice short-term in vivo carcinogenicity testing: lymphoma induction by benzo[a]pyrene, but not by TPA.

E mu-pim-1 transgenic mice are predisposed to develop lymphomas. Due to their low spontaneous tumour incidence and their increased sensitivity towards the lymphomagen ethylnitrosourea these mice may present an interesting model for short-term carcinogenicity testing. Here, we report on the further exploration of this transgenic mouse model with two additional carcinogens known to have, among others, the lymphohaematopoietic system as target, i.e. benzo[a]pyrene (B[a]P) and 12-O-tetradecanoylphorbol-13-acetate (TPA). B[a]P, given three times a week (by gavage) for 13 weeks at 4.3, 13 or 39 mg/kg body weight, resulted in a dose-related increase in lymphomas up to a 90% incidence in E(mu)-pim-1 mice during the observation period of 40 weeks. B[a]P also induced tumours of the forestomach within this observation period, though at a lower incidence and apparently equally effective in wildtype and transgenic mice. TPA, on the other hand, was unable to induce lymphomas (or tumours in any other organ) in either transgenic or wildtype animals within the observation period of 44 weeks, when applied dermally at the maximum tolerated dose of 3 microg/mouse, twice a week for 35 weeks. Molecular analysis showed that B[a]P-induced lymphomas in transgenic mice were of T-cell origin, 80% of which had elevated levels of c-myc expression. None of the lymphomas had increased N-myc expression and mutation analysis of the ras-gene family revealed a K-ras mutation in only one out of eight tumours investigated. Also, none of the lymphomas showed aberrant expression of p53 as determined by immunohistochemistry. It is concluded that the E mu-pim-1 mouse model will not be very suitable for short-term carcinogenicity testing in general: only genotoxic chemicals that have the lymphohaematopoietic system as target for carcinogenesis in wild-type mice, appear to be efficiently identified.

Induction of DNA adducts and mutations in spleen, liver and lung of XPA-deficient/lacZ transgenic mice after oral treatment with benzo[a]pyrene: correlation with tumour development.

We were interested to study the relationship between DNA lesions, DNA repair, mutation fixation, and tumour development. Therefore, mice harbouring lacZ reporter genes and being either wild-type or defective in the DNA excision repair gene XPA, were treated with the genotoxic carcinogen benzo[a]pyrene at an oral dose of 13 mg/kg b.w. (3 times/week). At different time points, i.e. 1, 5, 9 or 13 weeks after start of the oral administration, levels of BPDE-N2-dG adducts (the major formed DNA adduct by benzo[a]pyrene in mice), and lacZ mutation frequencies were measured both in target (spleen) and non-target (lung and liver) tissues. Both in wild-type and XPA-deficient mice, benzo[a]pyrene treatment resulted in increased BPDE-N2-dG adduct levels in all three tissues analysed. In XPA-deficient mice, BPDE-N2-dG adduct levels still increased up to 13 weeks of oral benzo[a]pyrene treatment, whereas in DNA repair proficient mice steady-state levels were reached after 5 weeks of treatment. After 13 weeks, the BPDE-N2-dG adduct levels observed in XPA-/- mice, were 2- to 3-fold higher than the steady state levels observed in XPA+/+ mice in the same tissues. Mutation frequencies in the lacZ reporter gene were the same in wild-type and XPA-deficient mice that were treated with the solvent only. Oral benzo[a]pyrene treatment resulted in an increase in mutation frequency in the lacZ marker gene in all three tissues, but this increase was most profound in the spleen. After 13 weeks of treatment, a 7-fold increase in lacZ mutation frequency was detected in the spleen of wild-type mice as compared to mutation frequencies in control mice. At the same time point, a 15-fold increase in lacZ mutation frequency was observed in the spleen of XPA-deficient mice. The data presented here show, that a defect in NER mainly results in enhanced mutation frequencies in lymphocytic cells after oral treatment with the genotoxic compound benzo[a]pyrene. Interestingly, as we established in a previously performed carcinogenicity assay, the same oral treatment with benzo[a]pyrene induced lymphomas residing in the spleen of XPA-deficient mice.

Cancer risk assessment for health care workers occupationally exposed to cyclophosphamide.

In the present study a cancer risk assessment of occupational exposure to cyclophosphamide (CP), a genotoxic carcinogenic antineoplastic agent, was carried out following two approaches based on (1) data from an animal study and (2) data on primary and secondary tumors in CP-treated patients. Data on the urinary excretion of CP in health care workers were used to estimate the uptake of CP, which ranged from 3.6 to 18 micrograms/day. Based on data from an animal study, cancer risks were calculated for a health care worker with a body weight of 70 kg and a working period of 40 years, 200 days a year (linear extrapolation). The life-time risks (70 years) of urinary bladder cancer in men and leukemias in men and women were found to be nearly the same and ranged from 95 to 600 per million. Based on the patient studies, cancer risks were calculated by multiplication of the 10-year cumulative incidence per gram of CP in patients by the estimated mean total uptake in health care workers over 10 years, 200 days a year. The risk of leukemias in women over 10 years ranged from 17 to 100 per million using the secondary tumor data (linear extrapolation). Comparable results were obtained for the risk of urinary bladder tumors and leukemias in men and women when primary tumor data were used. Thus, on an annual basis, cancer risks obtained from both the animal and the patient study were nearly the same and ranged from about 1.4 to 10 per million. In The Netherlands it is proposed that, for workers, a cancer risk per compound of one extra cancer case per million a year should be striven for ("target risk") and that no risk higher than 100 per million a year ("prohibitory risk") should be tolerated. From the animal and the patient study it appears that the target risk is exceeded but that the risk is still below the prohibitory risk.

Lipid peroxidation and protein thiol depletion are not involved in the cytotoxicity of N-hydroxy-2-acetylaminofluorene in isolated rat hepatocytes.

Freshly isolated rat hepatocytes were used to study the mechanism of cell death induced by N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Exposure to 1.0 mM N-OH-AAF resulted in more than 90% cell death (as measured by LDH leakage) of hepatocytes isolated from male rats within 6 hr. Only 36% of the hepatocytes isolated from female rats died within this period. When inorganic sulfate was omitted from the incubation medium, a 6 hr exposure to 1.0 mM N-OH-AAF resulted in only 40% cell death of male hepatocytes. These findings are in accordance with the sex difference and sulfation dependence of N-OH-AAF hepatotoxicity observed in the rat in vivo. N-OH-AAF decreased glutathione (GSH) in male hepatocytes in a concentration-dependent manner. This GSH consumption was only partly dependent on the presence of inorganic sulfate. No lipid peroxidation was observed during N-OH-AAF exposure; N-OH-AAF even prevented endogenous and diethyl maleate (DEM)-induced lipid peroxidation. No reduction of free protein thiol groups was found after exposure to N-OH-AAF, even after 75% cell death had occurred. A reduction of protein thiols after N-OH-AAF exposure was observed in GSH depleted hepatocytes (obtained by DEM plus vitamin E pretreatment). Under these conditions N-OH-AAF-induced cell death occurred earlier. Therefore, GSH protects against protein thiol depletion by N-OH-AAF in control cells. N-OH-AAF-induced cell death was preceded by a loss of intracellular ATP. It is concluded, therefore, that neither lipid peroxidation nor depletion of protein thiols, but possibly loss of intracellular ATP, is involved in the sulfation-dependent cytotoxic mechanism of N-OH-AAF in isolated rat hepatocytes.

Preventive action of thioethers towards in vitro DNA binding and mutagenesis in E. coli K12 by alkylating agents.

Thioethers are effective scavengers of electrophilic metabolites derived from the hepatocarcinogen N-hydroxy-2-acetylaminofluorene (van den Goorbergh et al., 1987). In this study 2 of these thioethers, 4-(methylthio)benzoic acid (MTB) and its methylester, methyl 4-(methylthio)benzoate (MMTB), have been tested for their ability to prevent in vitro DNA binding and mutation induction in E. coli K12 by the direct alkylating agents ethylnitrosourea (ENU), methylnitrosourea (MNU), ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS). In addition to MTB and MMTB, the thioether L-methionine (Met), and the thiols glutathione (GSH) and L-cysteine (Cys) were included for reasons of comparison. MTB was able to (partially) prevent DNA binding and mutation induction by ENU. However, this thioether was ineffective with EMS. DNA binding and mutagenesis by EMS were (partially) prevented by GSH and Cys, while these thiols could not prevent DNA binding and mutation induction by ENU. MMTB was unable to prevent mutation induction by these ethylating agents. With the methylating agents, similar effects of MTB were observed: MTB effectively prevented mutation induction by MNU while it was much less effective towards MMS. GSH and Cys were comparably effective as antimutagenic agents towards both methylating agents. Met was unable to prevent either DNA binding or mutation induction by these agents. Taken together, the results show that aromatic thioethers are able to trap genotoxic electrophiles derived from the nitrosoureas ENU and MNU, and may therefore act as potential anticarcinogens towards these agents, which are only poorly detoxified by GSH.

Extracellular calcium alleviates cell toxicity due to hepatotoxins that induce lipid peroxidation, but has no effect on toxins that do not cause lipid peroxidation. A study in isolated rat hepatocytes.

The effect of extracellular calcium on cell death, induced by hepatotoxins that induce lipid peroxidation [diethyl maleate (DEM), allyl alcohol (AA) and bromoisovalerylurea (BIU)] and hepatotoxins that do not induce lipid peroxidation [disulfiram (DSF), N-hydroxy-2-acetyl-aminofluorene (N-OH-AAF) and tetrahydroaminoacridine (THA)] was studied in freshly isolated rat hepatocytes. Extracellular calcium strongly delayed the onset of toxicity of DEM, AA and BIU as detected by lipid peroxidation, depletion of free protein thiol groups and cell death. This protective effect of calcium was decreased at higher concentrations of the toxic compounds. In contrast, no effect of calcium was observed on toxicity induced in the absence of lipid peroxidation by DSF, N-OH-AAF and THA. Addition of calcium was also without effect on the protein thiol depletion. These results indicate that calcium only alleviates cytotoxicity which is induced by thiol depletion resulting from lipid peroxidation. Cytotoxicity as a result of protein thiol depletion through disulfide formation is not affected by extracellular calcium.

The role of N-sulfation in the N-hydroxy-2-acetylaminofluorene-mediated outgrowth of diethylnitrosamine-initiated hepatocytes to gamma-glutamyltranspeptidase-positive foci in male rat liver.

The ability of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to promote the appearance of gamma-glutamyltranspeptidase-positive (GGT+) foci initiated by diethylnitrosamine (DEN) was studied in a slightly modified Solt and Farber protocol. This protocol consisted of the following treatments: initiation with a single dose of DEN followed by selection/promotion with several non-necrogenic doses of N-OH-AAF and partial hepatectomy. Treatment with N-OH-AAF resulted in a 25-fold increase of the liver volume occupied by GGT+ cells as compared to controls. The role of N-sulfation of N-OH-AAF in the GGT+ foci-selecting activity of N-OH-AAF was studied using the sulfotransferase inhibitor pentachlorophenol (PCP). Inhibition of the N-sulfation pathway with PCP during selection with N-OH-AAF resulted in a greater than 80% decrease in the volume occupied by GGT+ cells, without effects on the number of GGT+ foci generated with this protocol. Also, PCP reduced the number of so called oval and bile duct cells generated by the N-OH-AAF/partial hepatectomy treatment. It is concluded that N-sulfation of N-OH-AAF is responsible for the N-OH-AAF-mediated outgrowth of DEN-initiated hepatocytes to preneoplastic GGT+ foci.

Autoradiographic studies on in vivo covalent binding of N-hydroxy-2-acetylaminofluorene in rat liver containing gamma-glutamyltranspeptidase-positive foci. The effect of the sulfation-inhibitor pentachlorophenol.

The role of sulfation in the covalent binding of [ring-3H]-N-hydroxy-2-acetylaminofluorene (N-OH-AAF) in rat liver containing gamma-glutamyltranspeptidase-positive (GGT+) foci was studied in vivo by autoradiography. GGT+ foci were induced by initiation with diethylnitrosamine, followed by a selection protocol consisting of a 2-week exposure to 2-aminofluorene in the drinking water and a single administration of CCl4. Both surrounding ('normal') liver tissue and, to a lesser extent, GGT+ foci covalently bound N-OH-AAF, administered 10 days after selection. The sulfation inhibitor, pentachlorophenol (PCP), reduced the covalent binding of N-OH-AAF in cells surrounding the foci. However, PCP had no effect on binding of radiolabel in GGT+ foci. Thus, reduced sulfotransferase activity may contribute to the resistance of GGT+ preneoplastic lesions to carcinogen cytotoxicity. The results suggest also, that cells in GGT+ foci are able to bind N-OH-AAF covalently by a sulfotransferase-independent pathway.