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.

MxA protein--an interferon beta biomarker in primary progressive multiple sclerosis patients.

BACKGROUND AND PURPOSE: Interferon beta (IFNbeta) preparations have some effect on the progressive phase of multiple sclerosis (MS). This limited effect might be partially because of a certain number of IFNbeta non-responders. Myxovirus resistance protein A (MxA)--a marker of IFNbeta bioactivity--was correlated with the clinical response during an uncontrolled trial, investigating the safety of IFNbeta-1b in primary progressive (PPMS) patients. METHODS: Twenty PPMS were treated with IFNbeta-1b (s.c.) for 1 year. Blood samples were taken before and 1, 2, 3, 6, 9, 12, and 15 months after treatment initiation and MxA protein levels were measured. Patients were clinically evaluated by EDSS and the more sensitive Incapacity Status Scale (ISS) and stratified in a stable and a progressing group. RESULTS: Using ISS criteria, 11 patients remained stable and nine patients progressed during treatment. The mean area under the curve of log MxA levels during treatment were significantly higher in stable than in progressing patients (10.87 vs. 5.99; P = 0.002). CONCLUSION: A good biological response to IFNbeta might be associated with a better clinical effect of this drug and could be helpful in future clinical studies for early identification of treatment responders.

Development of a novel scoring system for identifying emerging chemical risks in the food chain.

The European Food Safety Authority (EFSA) is responsible for risk assessment of all aspects of food safety, including the establishment of procedures aimed at the identification of emerging risks to food safety. Here, a scoring system was developed for identifying chemicals registered under the European REACH Regulation that could be of potential concern in the food chain using the following parameters: (i) environmental release based on maximum aggregated tonnages and environmental release categories; (ii) biodegradation in the environment; (iii) bioaccumulation and in vivo and in vitro toxicity. The screening approach was tested on 100 data-rich chemicals registered under the REACH Regulation at aggregated volumes of at least 1000 tonnes per annum. The results show that substance-specific data generated under the REACH Regulation can be used to identify potential emerging risks in the food chain. After application of the screening procedure, priority chemicals can be identified as potentially emerging risk chemicals through the integration of exposure, environmental fate and toxicity. The default approach is to generate a single total score for each substance using a predefined weighting scenario. However, it is also possible to use a pivot table approach to combine the individual scores in different ways that reflect user-defined priorities, which enables a very flexible, iterative definition of screening criteria. Possible applications of the approaches are discussed using illustrative examples. Either approach can then be followed by in-depth evaluation of priority substances to ensure the identification of substances that present a real emerging chemical risk in the food chain.

Macrophages are eliminated from the injured peripheral nerve via local apoptosis and circulation to regional lymph nodes and the spleen.

The present study investigated the fate of macrophages in peripheral nerves undergoing Wallerian degeneration, especially their disappearance from the injured nerves after phagocytosis of axonal and myelin debris. Wallerian degeneration was induced in adult male C57Bl/6 mice by transecting the right sciatic nerve. Five days after transection, the male sciatic nerves were transplanted into female recipient mice by placing them exactly parallel to the host sciatic nerves. Nerves of the female recipient mice were also transected to induce breakdown of the blood-nerve barrier in the host animal. Apoptosis was assessed by morphological, immunohistochemical (activated caspase-3), and molecular (DNA fragmentation) methods in transplanted, recipient, and in control nerves. A subpopulation of macrophages within the degenerating nerves died locally by apoptosis in each experiment. The fate of the male macrophages within the transplanted nerves and the host organism was investigated by in situ hybridization with a Y-chromosome-specific DNA probe (145SC5). In situ hybridization specifically stained cells within the transplanted male nerve. Y-chromosome-positive cells were detected not only inside the transplanted nerve, but also inside the female host nerve, the perineurial tissue, the local perineurial blood vessels, draining lymph nodes and the spleen of the female host, suggesting hematogenous as well as lymphatic elimination of macrophages from the injured nerve. These data indicate that local apoptosis and systemic elimination via circulation to the local lymph nodes and the spleen are involved in the disappearance of macrophages from the injured peripheral nervous system.

Mutagenicity testing with transgenic mice. Part I: Comparison with the mouse bone marrow micronucleus test.

As part of a larger literature study on transgenic animals in mutagenicity testing, test results from the transgenic mutagenicity assays (lacI model; commercially available as the Big Blue(R) mouse, and the lacZ model; commercially available as the Mutatrade markMouse), were compared with the results on the same substances in the more traditional mouse bone marrow micronucleus test. 39 substances were found which had been tested in the micronucleus assay and in the above transgenic mouse systems. Although, the transgenic animal mutation assay is not directly comparable with the micronucleus test, because different genetic endpoints are examined: chromosome aberration versus gene mutation, the results for the majority of substances were in agreement. Both test systems, the transgenic mouse assay and the mouse bone marrow micronucleus test, have advantages and they complement each other. However, the transgenic animal assay has some distinct advantages over the micronucleus test: it is not restricted to one target organ and detects systemic as well as local mutagenic effects.

Assessment of mechanisms driving non-linear dose-response relationships in genotoxicity testing.

In genetic toxicology, risk assessment has traditionally adopted linear dose-responses for any compound that causes genotoxic effects. Increasing evidence of non-linear dose-responses, however, suggests potential cellular tolerance to low levels of many genotoxicants with diverse modes of action. Such putative non-linear dose-responses need to be substantiated by strong mechanistic data that identifies the mechanisms responsible for the tolerance to low doses. This can be achieved by experimental demonstration of cytoprotective mechanisms and by providing experimental support for the existence of tolerance mechanisms against low dose effects. By highlighting key experiments into low dose mechanisms, this review aims to clarify which mechanistic data are required to support the use of non-linear dose-response models in risk assessment. Such key experiments are presented and discussed for alkylating agents, oxidants, particulate matter, nucleoside analogues, topoisomerase inhibitors and aneugens and exemplify the use of gene knockout models or transgenic models as well as chemical modulators of key effectors of relevant pathways and their impact on dose-response relationships. In vitro studies are particularly valuable to elucidate mechanisms of low-dose protection or lack thereof, while in vivo experiments are most appropriate for deriving a safe dose. In order to evaluate the existence of non-linear dose-response relationships for genotoxicants, we suggest that careful attention should be given to the mode of genotoxic action, relevant biomarkers of exposure, as well as to the existence and impact of potential cytoprotective mechanisms like detoxifying metabolism and DNA repair.

The detection of human cytomegalovirus immediate early antigen in peripheral blood leucocytes.

Recently, Van der Bij et al. (1988) reported that active human cytomegalovirus (HCMV) infection could be diagnosed by the detection of HCMV immediate early antigen (IEA) directly in the peripheral blood leucocytes of renal transplant recipients. However, the indirect peroxidase technique used resulted in high background staining due to endogenous peroxidase activity and thus the detection of HCMV-IEA positive leucocytes, which are sometimes present in extremely low numbers, was not always reliable. In an attempt to solve this problem, we have evaluated the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique, immunogold-silver staining (IGSS), and several fixatives. Fixation with acetone: methanol 1:1 in conjunction with the APAAP technique proved to be the most successful method. In 155 blood samples obtained from 44 patients following renal transplantation and from three AIDS patients, the number of positive cells ranged between 1 and 700 out of 400,000 (median 2). In 23 samples from 11 patients (one AIDS patient) at least one positive cell was found. In this series there were no problems with the evaluation since strong positive signals were obtained without any background staining. We therefore recommend the use of this protocol for the rapid and reliable detection of HCMV-IEA in peripheral blood leucocytes.

The long persistence of CMV DNA in the blood of renal transplant patients after recovery from CMV infection.

A total of 30-50% of all renal transplant recipients undergo infections caused by human cytomegalovirus. With the introduction of ganciclovir and foscarnet for specific antiviral therapy there is an increasing demand for diagnostic tools that allow the early and rapid identification of CMV as the causative agent of the observed disease. We and others previously showed the direct detection of pp65 antigen in peripheral blood leukocytes to be an excellent marker for active cytomegalovirus infection. In order to establish whether the detection of CMV DNA by the polymerase chain reaction (PCR) supplies further information in this regard, we compared both methods. In 41 renal transplant patients the PCR assay yielded a sensitivity of 100% compared with 87.5% of the antigenemia assay. Specificities reached 67% and 92.5%, respectively. In 5 patients without both serological signs of infection and antigenemia, CMV DNA was also found. The duration of CMV DNA detection in PBL during active infection was significantly longer than antigenemia. Even after successful treatment of symptomatic CMV disease, DNA was present for a period of weeks without any relapse of disease. In contrast, antigenemia disappeared after antiviral therapy and reappeared only in one patient with relapse of CMV disease. We conclude that PCR offers no advantages over antigen detection in monitoring for CMV infections after renal transplantation.

Pentoxifylline, a phosphodiesterase inhibitor, induces immune deviation in patients with multiple sclerosis.

The outcome of immune responses can be predicted by the lymphokine production pattern of the participating cells. Cytokines of the T helper type 1 (Th1) cells mediate inflammatory responses and delayed-type hypersensitivity (DTH), whereas Th2-like T cells predominantly produce cytokines, which stimulate antibody production by B cells. Immunoregulatory therapy of autoimmune diseases with unknown antigens may be achieved by inhibiting the production of inflammatory cytokines and induction of protective cytokines of Th2-like T cells. To determine the immunoregulatory capacity of the phosphodiesterase inhibitor pentoxifylline (PTX), which is known to suppress the production of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), this drug was used in mitogen and antigen-stimulated lymphocyte cultures as well as in patients with multiple sclerosis. PTX significantly decreased TNF-alpha and interleukin-12 (IL-12), whereas it increased IL-4 and IL-10 production. In addition, PTX inhibited cell proliferation, which was associated with a marked reduction in CD25 (IL-2 receptor alpha-chain) and CD54 (intercellular adhesion molecule-1; ICAM-1) expression. Increasing doses of PTX significantly reduced TNF-alpha and IL-12 mRNA expression of blood mononuclear cells, but increased IL-4 and IL-10 expression in eight patients with relapsing-remitting multiple sclerosis. These results indicate that PTX modulates immune reactions favouring a Th2-like response and may therefore be useful for the treatment of autoimmune diseases with a dominant Th1-like T cell response.

Analysis of interleukin-4 receptor alpha chain variants in multiple sclerosis.

A recent candidate gene study employing microsatellite markers suggested a possible linkage of multiple sclerosis (MS) with the interleukin-4 receptor (IL4R) gene. Consequently, we investigated the association of different IL4R variants with MS in 341 German MS patients and 305 healthy controls. Analysis of the first 100 MS patients for six IL4R variants showed an increased frequency of the R551 variant in MS patients versus healthy controls and carriage of the same IL4R variant was weakly associated with myelin oligodendrocyte glycoprotein (MOG) autoantibody production. However, further analysis of all 341 MS patients did not confirm the finding that this IL4R variant represents a general genetic risk factor for MS but revealed an increased frequency of the R551 variant in MS patients with primary progressive MS (PPMS, n=48) as compared to patients with relapsing remitting MS or secondary progressive MS (RR/SPMS n=284; P=0.005 for genotype differences) and to 305 healthy controls (P=0.001 for genotype differences). This association was statistically independent of the presence of the well-known MS susceptibility allele HLA-DRB1*15. After correction for multiple comparisons only the genotype differences between PPMS patients and healthy controls remained statistically significant. These results indicate, that the IL4R variant R551 may influence the genetic predisposition for PPMS but does not represent a general genetic risk factor for MS.

The macrophage activity marker sCD14 is increased in patients with multiple sclerosis and upregulated by interferon beta-1b.

The soluble form of the CD14 molecule (sCD14), a macrophage activity marker, was measured in the plasma of 17 patients with primary progressive multiple sclerosis (PPMS) and 20 patients with relapsing remitting MS (RRMS). In patients with PPMS, sCD14 levels were determined before and after treatment with interferon beta (IFNB). In both PPMS and in RRMS, sCD14 levels were significantly elevated compared to healthy controls. In patients with PPMS, sCD14 levels increased significantly during the first 3 months of IFNB therapy, then slightly decreased, but still remained elevated compared with levels before therapy. Therefore, the elevated sCD14 levels may be a marker in evaluating biological response to IFNB therapy.

c-fos gene expression in rat liver is induced by phenobarbital.

In the present study we investigated c-fos expression in rat livers, that was initiated with the three arylamines, 2-acetylaminofluorene, 2-acetylaminophenanthrene and trans-4-acetylaminostilbene. The tumor promoter phenobarbital was applied chronically for 26, 52 and 100 weeks. Gene expression, determined by the mRNA level, and FOS protein were increased after 52 weeks of treatment in arylamine initiated as well as in phenobarbital only treated animals. Expression of c-fos seems to be a phenobarbital induced effect that is independent of additional initiator treatments. This finding was supported by immunohistochemical studies demonstrating increased FOS levels to be localized around the central vein. The results indicate that phenobarbital, a widely used tumor promoter, induces c-fos expression. In addition, we demonstrated enhanced FOS in GST-P-positive foci and in tumors.

Early initiating and promoting effects in 2-AAF-induced rat liver carcinogenesis: an immunohistochemical study.

2-Acetylaminofluorene (2-AAF) is a complete carcinogen in rat liver. To investigate the specific properties, that distinguish 2-AAF from incomplete carcinogens, rats were fed 0.02% AAF in the diet for 6, 12, 16 weeks and some indicators of genotoxic and chronic toxic effects were studied immunohistochemically. GST-P, a marker for single initiated cells and preneoplastic foci, was induced in response to 2-AAF exposure. The effects were slight after 6 weeks of feeding, after 12 weeks GST-P-positive preneoplastic foci were present. The proto-oncogenes c-fos and c-jun are induced by several tumor promoters. In the present study c-FOS protein levels were increased in all 2-AAF treated animals at early stages not only in preneoplastic foci. However, all GST-P-positive foci were also c-FOS-positive. Surprisingly c-JUN was not enhanced in GST-P positive foci. It was comparatively expressed in hepatocytes and bile duct cells in all animals. We did not observe any immunolabeling for p53, either in preneoplastic foci or in hepatocytes from treated animals. A significant increase of apoptoses was noted in the whole liver lobule but also gathered in groups in the periportal area. The results support our proposal that oxidative stress and energy impairment in the mitochondria of periportal hepatocytes trigger morphological alterations in the rat liver.

New insights into carcinogenesis of the classical model arylamine 2-acetylaminofluorene.

2-Acetylaminofluorene (AAF) is a complete carcinogen in rat liver. The genotoxic effects of reactive metabolites are considered necessary but not sufficient to explain tumor formation. An overview is given of an AAF-feeding experiment designed to demonstrate early effects, preceding the development of enzyme-altered foci to support the hypothesis that toxic effects lead to a cirrhosis-like transformation as a prerequisite for the expansion of initiated foci and how those effects influence the dose-time-response relationship of tumor formation. Male Wistar rats were fed 0.005, 0.01, 0.02, 0.04 and 0.08% AAF in the diet for 2, 4, 8, and 16 weeks. GST-P-positive foci developed more than proportionately only at 16 weeks. As a first sign of morphological alterations the number of apoptoses increased (2 weeks), the proliferation rate followed with some delay and was maximal at 4 weeks. The most sensitive parameter for adaptive responses was the inhibition of the mitochondrial permeability transition, studied ex vivo. All parameters increased dose-dependently at low doses. A threshold could not be detected, but effects developed much more gradually with the lowest, non-toxic dose. The situation of massive development of foci observed with the higher doses at 16 weeks was not reached. Apoptosis and proliferation rate reach a plateau between 4 and 8 weeks with some of the doses indicating a period in which some balance between adaptation and stress response exists.

The role of nongenotoxic mechanisms in arylamine carcinogenesis.

The growth of preneoplastic nodules during the feeding of a carcinogenic 2-acetylaminofluorene (2-AAF) regimen is preceded by several alterations in the physiologic homeostasis. Many of these alterations can be considered adaptive responses to the drug exposure. One property of AAF could be identified that clearly distinguishes this complete rat liver carcinogen from at least two other, incomplete rat liver carcinogens. Highly specific redox cycling in mitochondria was demonstrated in vitro, and this observation could well contribute an explanation of the morphologic and histochemical observations in vivo. It is emphasized that nongenotoxic effects may play an important role in the generation of tumors by genotoxic carcinogens.

Dose response of early effects related to tumor promotion of 2-acetylaminofluorene.

The genotoxic effects of 2-acetylaminofluorene (AAF) alone cannot explain the formation of rat liver tumors. It has been proposed that mitochondrial effects are associated with its tumor-promoting properties. These mitochondrial effects are thought to trigger apoptosis and regenerative proliferation, which alters the liver lobule in a cirrhosis-like manner. A situation is generated which favors the growth of initiated cells. To test this sequence of events, the dose dependence of early effects with time was studied. Male Wistar rats received 50, 100, 200, 400, or 800 ppm AAF in the diet and the following endpoints were analyzed at 2, 4, 8, and 16 weeks of feeding: apoptotic cell death, cell proliferation, GST-P-positive foci (placental form of glutathione S-transferase), and morphological alterations. Hydrolyzable hemoglobin adducts were used as a dosimeter for metabolic activation after 2 weeks of feeding. The hemoglobin adduct levels increased linearly with dose. With the conventional carcinogenic concentration of 200-ppm AAF in the diet, the number of apoptoses increased first, predominantly in the periportal area (2 weeks). Cell proliferation followed with some delay (4 weeks). This reflects regenerative tissue response and not the growth of initiated cells, because the number of enzyme-altered foci was still extremely low at that time. Foci developed only later when the morphology had changed. With 50 ppm AAF in the diet, a no-effect level had not been reached for any of the endpoints, but foci developed much more gradually than with higher doses. Unexpectedly, the proliferative response stabilized at 8 weeks with a labeling index of 12-17, with all AAF concentrations. The observed sequence of events supports the hypothesis. It is concluded that (1) The proliferation of initiated cells-defined as promotable cells-is largely determined by the cellular environment, such as morphologically altered liver. (2) The morphological alterations in rat liver result from imperfect regeneration from toxic effects. (3) Imperfect regeneration results from limitations in the possibilities to adapt to chemical stress. (4) If these limits are overwhelmed and morphology has changed to a certain extent, preneoplastic foci develop; this occurs much faster, at least, than without these changes.

Gene polymorphism at position -308 of the tumor-necrosis-factor-alpha (TNF-alpha) in multiple sclerosis and it's influence on the regulation of TNF-alpha production.

Tumor-necrosis-factor alpha (TNF-alpha) is a major mediator of the inflammatory immune response and may play an important role in the pathogenesis and progression of Multiple Sclerosis (MS). Increased TNF-alpha levels of cerebrospinal fluid (CSF) and peripheral blood were found in patients with chronic progressive MS and patients with acute relapses, but not in the stable form of the disease. Considering the association of different TNF-alpha alleles with diverse autoimmune diseases we sequenced the TNF-alpha promotor region (-674 to +201) of 23 patients with relapsing/remitting MS, of 27 patients with chronic progressive MS (21 patients had primary progressive course and six patients had a secondary progressive course) and of 22 healthy controls, who had no history of MS in their families. In three of 21 patients (14%) with primary chronic progressive MS a homozygous point-mutation at position -308 could be demonstrated where guanine (G) was substituted by adenosine (A). This mutation could neither be detected in patients with relapsing/remitting MS nor in healthy controls. However, 40% of the patients with relapsing/remitting MS and 43% of the primary chronic progressive MS patients were heterozygous at position -308 for G/A, whereas only 32% of healthy controls showed this heterogeneity. The genetic variations were demonstrated by polymerase chain reaction (PCR)-amplification of the TNF-alpha promotor-region and consecutive direct automatic sequencing. Functional analysis of the promoter region using the chloramphenicol-acetyltransferase (CAT) assay revealed spontaneous production with the homozygous mutation at -308 only.

Central nervous system TNFalpha-mRNA expression during rabbit experimental pneumococcal meningitis.

In pneumococcal meningitis inflammatory mediators such as tumor necrosis factor alpha (TNFalpha) are produced in large quantities and play a major role in pathogenesis. It is not known exactly which cells produce these mediators during infection. We investigated the localisation of TNFalpha-mRNA in the central nervous system (CNS) by in situ hybridisation during experimental Streptococcus pneumoniae meningitis. TNF-positive cells were detected only in inflammatory infiltrates within the meninges. Cells within the brain parenchyma and the choroid plexus were completely negative. After monocyte depletion, no TNFalpha-mRNA positive cells were detected in the CNS. These findings suggest that TNFalpha in pneumococcal meningitis is produced in the CNS mainly by blood-derived, infiltrating monocytes.

Inflammatory CNS demyelination: histopathologic correlation with in vivo quantitative proton MR spectroscopy.

BACKGROUND AND PURPOSE: The mechanisms behind the demyelination that is characteristic of multiple sclerosis (MS) are still poorly understood. The purpose of this study was to compare immunopathologic findings in demyelinating lesions of three patients with in vivo assessments obtained by quantitative proton MR spectroscopy (MRS). METHODS: Between four and seven stereotactic needle brain biopsies were performed in three young adults with diagnostically equivocal findings for MS. Axonal density, gliosis, blood brain-barrier breakdown, and demyelinating activity of lesions were determined. Combined MR/MRS studies were performed (T1-weighted fast low-angle shot and single-voxel stimulated-echo acquisition mode), and absolute metabolite levels were obtained with a user-independent fitting routine. Metabolite control values were obtained from a group of age-matched healthy volunteers (n = 40, age range, 20-25 years old). Alterations of metabolite levels of control subjects were considered significant when exceeding two standard deviations. RESULTS: There were parallel decreases of N-acetylaspartate (21%-82%) and reductions of axonal density (44%-74%) in demyelinating plaques. Concomitant increases of choline (75%-152%) and myo-inositol (84%-160%) corresponded to glial proliferation. Elevated lactate was associated with inflammation. CONCLUSION: The present data suggest that in vivo MRS indicates key pathologic features of demyelinating lesions.

The structure and function of the H-ras proto-oncogene are not altered in rat liver tumors initiated by 2-acetylaminofluorene, 2-acetylaminophenanthrene and trans-4-acetylaminostilbene.

Liver tumors were generated in Wistar rats in an initiation-promotion experiment. 2-Acetylaminofluorene (AAF), 2-acetylaminophenanthrene (AAP), and trans-4-acetylaminostilbene (AAS) were administered to newborn animals as initiators, and phenobarbital as a promoter was added to the drinking water after weaning. Livers were examined after 26, 52, 78, and 104 weeks. Tumors were present in all groups except for at the first time point. The potency of the initiators decreased in the order AAS > AAP > AAF. DNA from tumors of all groups and of control livers was analyzed for mutations in the H-ras gene, but no mutations could be found. The sequence of almost the entire H-ras gene was determined and was compared to other H-ras genes. There are some differences with the sequence in other rat strains, particularly in intron D containing the alternative splicing site. The expression of the H-ras gene has also been studied by various methods in enzyme altered foci and tumors, but no alterations could be found. It is, therefore, concluded that structural of functional alterations of this proto-oncogene are not involved in the generation of liver tumors in Wistar rats by the three genotoxic arylamines.