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.

Histopathological effects of chronic aqueous exposure to bis(tri-n-butyltin)oxide (TBTO) to environmentally relevant concentrations reveal thymus atrophy in European flounder (Platichthys flesus).

Although the use of tributyltin in antifouling paints has been banned, this compound is still a serious pollutant of the marine environment. This paper describes a unique study in which European flounder (Platichthys flesus) were chronically (8 months) exposed to bis(tri-n-butyltin)oxide (TBTO) in the water under controlled laboratory conditions. Residue levels in selected tissues (liver, muscle) and general health status indices were measured and the effects on several organs (gills, liver, mesonephros, ovary/testis, spleen, and gastrointestinal tract) were examined histopathologically. Additionally, morphometric analysis of the thymus was performed. The major finding is that exposure of flounder to 5 microg TBTO/l over a period of 8 months, resulting in body burdens comparable to high field levels, induced significant reduction of thymus volume, possibly affecting immunocompetence of the animals. Chronic exposure of European flounder to tributyltin is therefore likely to affect the general health status of this species in heavily polluted aquatic environments.

Long-term exposure of European flounder (Platichthys flesus) to the flame-retardants tetrabromobisphenol A (TBBPA) and hexabromocyclododecane (HBCD).

Tetrabromobisphenol A (TBBPA) and hexabromocyclododecane (HBCD) are widely used flame retardants that have increasingly been found as contaminants in the aquatic environment. In the present study, European flounder (Platichthys flesus) were chronically exposed to TBBPA; (105 days) and HBCD (78 days), in a wide range including environmentally relevant concentrations. TBBPA was administered via the water, whereas HBCD was administered in food and sediment, or in sediment alone. Chemical analysis of muscle showed an average increase in internal concentrations of approximately two orders of magnitude for both compounds tested. Animals exposed to HBCD via sediment alone (8000 microg/g total organic carbon, TOC) showed a proportional increase of alpha-HBCD in muscle compared to animals exposed via food and sediment. In both studies, exposure to the test compounds did not affect general health and toxicity parameters (behavior, survival, growth rate, relative liver and gonad weight). Hepatic microsomal enzyme activities (TBBPA: EROD; HBCD: EROD, PROD, and BROD) were not induced by any of the tested chemicals. Aromatase activity in male gonads showed a mild increase with rising TBBPA levels. There were no morphological and immunohistochemical indications for increased production of the yolk precursor protein vitellogenin (VTG) in animals exposed to TBBPA and HBCD; immunochemical analysis of plasma VTG levels showed no dose response in animals exposed to TBBPA. In animals exposed to TBBPA, levels of the thyroid hormone thyroxin (T(4)) increased with internal concentrations of the test compound, possibly indicating competition of TBBPA for plasma protein binding. Triiodothyronin (T(3)) levels were not affected and histology showed no signs of altered thyroid gland activity. Other organs investigated (liver, gills, kidney, skin, and gonads) revealed no histological changes related to TBBPA or HBCD exposure. Overall, the present results indicate limited endocrine effects of these widely used flame retardants in a test species representative of European estuaries at environmentally relevant exposure levels and at internal levels up to 4300 ng TBBPA/g wet weight, and 446 microg HBCD/g lipid weight in flounder muscle.

Toxicity of tetrabromobisphenol A (TBBPA) in zebrafish (Danio rerio) in a partial life-cycle test.

Toxicological effects of the widely used flame retardant, tetrabromobisphenol A (TBBPA) were assessed in a partial life-cycle test with zebrafish (Danio rerio). Exposure of adult fish during 30 days to water-borne TBBPA in nominal concentrations ranging from 0 (control) to 1.5 microM was followed by exposure of the offspring in early life stages up to 47 days posthatching (dph) to the same concentrations. Adults exposed to 3 and 6 microM showed severe disorientation and lethargy shortly after beginning of exposure and were euthanized. Because semistatic exposure resulted in fluctuating water concentrations, pooled fish samples were chemically analyzed for internal dose assessment. Egg production was decreased in fish exposed to TBBPA concentrations of 0.047 microM and higher, and a critical effect level of 7.2 microg/g lipid with a lower 5% confidence limit of 3.9 microg/g lipid for 50% decreased egg production was calculated. Histology of adult ovaries indicated a relative increase of premature oocytes in two surviving females exposed to 1.5 microM. Hatching of TBBPA-exposed larvae was decreased except in animals exposed to 0.375 microM. In the highest exposure concentration, early posthatching mortality was high (81%) in larvae and the surviving juveniles showed a significant predominance of the female phenotype. Exposure of eggs from control parents up to 6 microM TBBPA resulted in increasing malformation and pericardial fluid accumulation from 1.5 microM; at higher concentrations, all embryos failed to hatch. The presented results indicate decreased reproductive success in zebrafish at environmentally relevant TBBPA concentrations.

Aquatic toxicology: opportunities for enhancement through histopathology.

This paper briefly reviews the application of histopathology as aninstrument or endpoint in toxicity studies in fish. For long this has been applied rather occasionally in (regulatory) toxicology, and was mainly of interest in fundamental studies and limited carcinogenicity experiments. However, nowadays there are various incentives that ask for the application of pathology, such as field monitoring of pollution effects, the wish for optimal use and lower species of laboratory animals, the availability of modern histology techniques, and insight and interest in mechanistic data. This is timely illustrated by the current broad interest in endocrine disrupting pollutants-a threat mainly in the aquatic environment-where histopathological organ and tissue changes in intact sentinel fish species provide pivotal diagnostic and mechanistic features.

Toxicity of PCB-126 in European flounder (Platichthys flesus) with emphasis on histopathology and cytochrome P4501A induction in several organ systems.

A series of experiments was set up to elucidate the effects of pollution on marine and estuarine fish health, since the European flounder (Platichthys flesus) has shown a relatively high prevalence of (pre)neoplastic liver lesions and lymphocystis virus disease in Dutch coastal and estuarine waters. The hypothesis of a causal relationship between pollution and the above-mentioned diseases was supported by results from semi-field experiments. Therefore several laboratory experiments were carried out to substantiate causality further and to identify the xenobiotics that may play a major role in the field. The present study focuses on polychlorinated biphenyls (PCBs). European flounders (Platichthys flesus) were orally exposed to a single dose of 0, 0.5, 5 or 50 mg PCB-126/kg body weight under controlled laboratory conditions. The effects on liver, gills, gastrointestinal tract, gonads, spleen and mesonephros were examined histologically after 16 days. Induction and localization of cytochrome P4501A (CYP1A) immunoreactivity, and effects on hepatocyte proliferation were visualized immunohistochemically. Effects on thymus size were examined by morphometric analysis of serial sections. Three out of five animals of the highest dose group showed haemorrhages in the fins and tail after 16 days. All animals showed reduced activity in the later stages of the experiment, and some animals of the highest dose group discontinued feeding 14 days after exposure. Strong and exposure-related induction of CYP1A immunoreactivity was noted in hepatocytes, endothelium in all organs examined, and epithelium of the digestive tract and mesonephros at PCB-126 levels of 0.5, 5 and 50 mg/kg. In addition, the strong induction of CYP1A immunoreactivity in a distinct population of haematopoietic cells in the mesonephros and in circulating blood is remarkable, and has not been described previously in other fish species. Furthermore, a morphometrically determined significant reduction in relative thymus size was noted in animals exposed to 50 mg PCB-126/kg. Although the functional implications for the immune system of this reduction need to be further investigated, an impact on the specific resistance against infectious diseases as observed in the field, e.g. viral lymphocystis disease, is not implausible. In addition, a significant increase in absolute liver weight, in hepatosomatic index, and in number of proliferating hepatocytes [measured as immunoreactivity against proliferating cell nuclear antigen (PCNA)] was noted in animals of the highest dose group. From these findings we suppose that PCB-126 (and related chemicals) may play a role in the promotion of tumour development in the liver of European flounders as observed in the field. The results of the present experiment show relatively stronger effects than effects previously reported from experiments with TCDD, suggesting that the TEF of 0.005 assigned to PCB-126 from early life stage mortality experiments in rainbow trout (Oncorhynchus mykiss), underestimates the toxic potential of PCB-126.

Toxicology of environmental chemicals in the flounder (Platichthys flesus) with emphasis on the immune system: field, semi-field (mesocosm) and laboratory studies.

European flounder (Platichthys flesus) has shown an increased prevalence of liver tumors and lymphocystis disease (a viral infection) that correlated with pollution in field research in Dutch coastal and estuarine waters. Semi-field or mesocosm experiments confirmed the supposed causality. Although these types of research are highly relevant for the feral population, laboratory experiments are necessary to establish causal relationships between specific chemical pollutants and disease. Therefore, the effects on flounder of some of the potentially causative chemicals such as benzo[a]pyrene (BaP), dimethyl-benz[a]anthracene (DMBA), 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), 3,3',4,4',5 pentachlorobiphenyl (PCB-126), and bis(tri-n-butyltin)oxide (TBTO) were examined in several laboratory experiments. These effects were evaluated using general toxicological parameters and histopathology. For immune function assessment, attempts to develop an infection model with the lymphocystis virus were made, but appeared unsuccessful and immune function tests are not fully operational at the moment. Flounder has been successfully maintained and exposed to toxic substances in captivity in our laboratory. Short-term aqueous exposure to high levels of BaP or DMBA did not induce marked effects under our experimental conditions. Results of oral exposure of flounder to low levels of TCDD, PCB-126 or harbor sludge extract show significant induction of cytochrome P4501A (CYP1A) in hepatocytes. Oral exposure to high levels of TCDD or PCB-126 also significantly induced CYP1A immunoreactivity in epithelium in mesonephros and digestive tract and in endothelium in several organs. Remarkable was the induction of CYP1A in a distinct population of mononuclear cells in the mesonephros. Moreover, oral exposure to TCDD resulted in an increased mitotic activity and an increase of the hepatosomatic index in the 20 and 500 microgram TCDD/kg group respectively. Therefore, exposure to TCDD and related substances may promote the development of liver tumors in the field. Exposure to PCB-126 also significantly reduced the relative thymus volume, but other results indicate that flounder is relatively insensitive to this type of chemicals. Short-term aqueous exposure of flounder to TBTO, in concentrations that were in the same order of magnitude as upper TBT levels measured in the field, caused mortality after 7-12 days associated with gill lesions, and induced reduction of the non-specific resistance and decrease of the relative thymus volume. From these results we therefore conclude that TBTO might play a causal role in, for instance, increased prevalence of lymphocystis virus infections in the field

Assessment of side effects induced by injection of different adjuvant/antigen combinations in rabbits and mice.

We evaluated the side effects induced by injection of Freund's adjuvant (FA) and alternative adjuvants combined with different antigens. Rabbits and mice were injected subcutaneously, intramuscularly (rabbits) and intraperitoneally (mice) with different adjuvants (FA, Specol, RIBI, TiterMax, Montanide ISA50) in combination with several types of antigens (synthetic peptides, autoantigen, glycolipid, protein, mycoplasma or viruses). The effects of treatment on the animals' well-being were assessed by clinical and behavioural changes (POT and LABORAS assays) and gross and histopathological changes. In rabbits, treatment did not appear to induce acute or prolonged pain and distress. Mice showed behavioural changes immediately after (predominantly secondary) immunization. Injection of several adjuvant/antigen mixtures resulted in severe pathological changes, depending on adjuvant, type of antigen, animal species used and route of injection. Both rabbits and mice showed pathological changes ranging from marked to severe after injection of FA, and ranging from minimal to marked after Specol and Montanide injections. Pathological changes after RIBI injections were severe in rabbits, though slight in mice. After TiterMax injections, pathological changes were moderate in rabbits, though severe in mice. In conclusion, injection of FA according to present guidelines resulted mostly in severe pathological changes, whereas only very few clinical and behavioural signs indicated prolonged severe pain. Our findings indicate that Montanide ISA50 and Specol induce acceptable antibody titres, and cause fewer pathological changes than FA. Thus they are effective alternatives to FA.

XPA-deficiency in hairless mice causes a shift in skin tumor types and mutational target genes after exposure to low doses of U.V.B.

Xeroderma pigmentosum (XP) patients with a defect in the nucleotide excision repair gene XPA, develop tumors with a high frequency on sun-exposed areas of the skin. Here we describe that hairless XPA-deficient mice also develop skin tumors with a short latency time and a 100% prevalence after daily exposure to low doses of U.V.B. Surprisingly and in contrast to U.V.B.-exposed repair proficient hairless mice who mainly develop squamous cell carcinomas, the XPA-deficient mice developed papillomas with a high frequency (31%) at a U.V. dose of 32 J/m2 daily. At the highest daily dose of 80 J/m2 mainly squamous cell carcinomas (56%) and only 10% of papillomas were found in XPA-deficient hairless mice. p53 gene mutations were examined in exons 5, 7 and 8 and were detected in only 3 out of 37 of these skin tumors, whereas in tumors of control U.V.B.-irradiated wild type littermates this frequency was higher (45%) and more in line with our previous data. Strikingly, a high incidence of activating ras gene mutations were observed in U.V.B.-induced papillomas (in 11 out of 14 tumors analysed). In only two out of 14 squamous cell carcinomas we found similar ras gene mutations. The observed shift from squamous cell carcinomas in wild type hairless mice to papillomas in XPA-deficient hairless mice, and a corresponding shift in mutated cancer genes in these tumors, provide new clues on the pathogenesis of chemically- versus U.V.B.-induced skin carcinogenesis.

The role of UV-B light in skin carcinogenesis through the analysis of p53 mutations in squamous cell carcinomas of hairless mice.

Mutation spectra of the p53 gene from human skin carcinomas have been connected to solar UV radiation. For comparison we have characterized the mutation spectrum of the p53 gene in a very large sample of squamous cell carcinomas from hairless mice induced with UV of wavelength 280-320 nm (UV-B), which have substantiated the mutagenic effects of UV-B radiation in vivo. Tumors from hairless mice, random bred SKH:HR1 as well as inbred SKH:HRA strains, which are analyzed for mutations in the conserved domains of the p53 protein present a very specific mutation spectrum. The observed mutation frequency after chronic UV-B radiation in the p53 gene ranged from 54% (SKH-HRA) to 73% (SKH-HR1) among the 160 tumors analyzed. Over 95% of the mutations were found at dipyrimidine sites located in the non-transcribed strand, the majority were C-->T transitions and 5% were CC-->TT tandem double mutations. Four distinct UV-B mutation hot spots have been identified for the first time: two major ones at codons 267 (33%) and 272 (19%) and two minor ones at codons 146 (10%) and 173 (4%). The codon 267 hot spot consists of a CpG preceded by a pyrimidine, which confirms in vivo an important role for this UV-B mutable site in UV-B-induced skin tumors that is not found in other types of mouse tumors. Comparison with mutation spectra from human skin carcinomas fully validates the merits of the hairless mouse model for studying the molecular mechanisms of skin carcinogenesis. For example, the murine hot spot at codon 272 does have a full equivalent in human skin carcinomas. In contrast, the human equivalent of the murine codon 267 lacks the dipyrimidine site and therefore fails to be a pronounced hot spot in human skin carcinomas; however, this site is one of the major hot spots in human internal cancers (evidently not induced by UV radiation but probably by deamination of the 5 methyl cytosine).

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.

Low incidence of p53 mutations in UVA (365-nm)-induced skin tumors in hairless mice.

Mutations with clear "UVB fingerprints" have been observed in the p53 gene of human nonmelanoma skin tumors and of experimentally UVB-induced murine skin tumors. Although UVA (315-400 nm) radiation is also a complete carcinogen, its contribution to sunlight-induced mutagenesis remains poorly characterized. There is experimental evidence that the production of reactive oxygen species plays a more dominant role with long-wave UVA than with UVB radiation. We have induced skin tumors (n = 42) in hairless SKH:HR1 mice (n = 14) by daily exposure to long-wave UVA (365-nm) radiation. The incidence of p53 alterations in these tumors is low compared to UVB-induced tumors; positive staining for the p53 protein was observed in only 50% of the tumors, and less than 15% of the tumors showed a mutation in one of the exons 5, 7, or 8 of the p53 gene. The pattern of p53 staining was more irregular and less dense compared to UVB, and the mutations (all C-->T) were mainly (six of seven) located at codon 267. Besides a general p53 hotspot, this codon is also the main hotspot for UVB-induced skin tumors in these mice. No mutations specific for UVA, ie., mutations specific for reactive oxygen species, could be detected.

Risk assessment on the carcinogenic potential of hybridoma cell DNA: implications for residual contaminating cellular DNA in biological products.

The aim of this study was to evaluate the possible carcinogenic potential of residual DNA derived from immortalized and possibly tumorigenic cell lines due to activated oncogenic sequences (oncogenes). These cell lines have been used for the production of biologicals, i.e. monoclonal antibodies, lymphokines and vaccines. The authors used hybridoma DNA as a first model. For this reason experiments in two species were performed, namely in 3-4 week-old female Balb/c mice and newborn Riv:TOX rats. Doses of 250 micrograms DNA, derived from Balb/c hybridoma cells, were injected subcutaneously (s.c.) in 200 mice. These mice also received a s.c. injection of the solvent only (TE buffer) at another site of the back skin (negative control for local tumour development). An additional group of 50 mice was treated intraperitoneally (i.p.) with the solvent only to serve as a negative control group for possible systemic tumorigenic effects. Doses of 5 micrograms plasmid pPy1 DNA, containing the entire Polyoma virus genome, served as positive control and were injected s.c. and i.p. in 20 and 50 mice, respectively. Doses of 50 micrograms hybridoma DNA or 5 micrograms pPy1 DNA were injected s.c. in rats too, using nine animals per group. During the experiment, animals were observed weekly, especially for the occurrence of subcutaneous tumours at the injection sites. The mouse study was terminated after more than 2 years, the rat study after 1 year. Gross necropsy was performed on all animals and histopathological examination of grossly suspected neoplastic lesions was performed. In the mouse experiment, tumour development at the s.c. injection site of the DNA was observed in one out of 20 animals in the pPy1-treated positive control group (neurofibrosarcoma) and one out of 200 animals in the hybridoma DNA-treated group (haemangioma-like lesion). Tumour development at or near the s.c. injection site of the solvent only was observed in two out of 200 animals. In the rat study none out of nine hybridoma DNA-treated rats developed tumours at the injection site, while three out of nine rats of the positive control group, injected with the pPy1 DNA, showed local tumour development (benign and malignant soft tissue tumours.) It is concluded that, at the high dose and numbers of animals tested, parenteral administration of hybridoma DNA does not induce local tumour development. Furthermore, no indications were found for systemic carcinogenic potential of the hybridoma DNA used. Based on a worst case approach of our data, the oncogenic risk of 100 pg residual DNA was estimated to be 2 x 10(-9), a value intermediate of the estimations of the WHO (1987) and the Dutch Health Council (1988) 5 x 10(-11) and 2 x 10(-7), respectively. Therefore, it is unlikely that the risk of 100 pg of DNA derived from other immortalized cell lines will exceed the level of generally accepted cancer risk of 10(-6).

Skin and liver diseases induced in flounder (Platichthys flesus) after long-term exposure to contaminated sediments in large-scale mesocosms.

Disease development in flounder (Platichthys flesus) was studied over a period of 3 years in three large mesocosms (40 m x 40 m x 3 m). Two of the mesocosms contained clean sand and the third, sharing a common water circulation with one of the clean-sand mesocosms, was stocked with contaminated dredged spoil. In this way, one of the clean-sand mesocosms was indirectly polluted via the water phase, and analysis of contaminant concentrations in sediments and flounder tissues showed that it had a status intermediate between the other two. Random samples of the flounder populations from the indirectly polluted and reference mesocosms were examined every 2 months for epidermal diseases (lymphocystis, skin ulcers, fin rot) and then released. In addition, every 6 months, random samples of fish from all three mesocosms were sacrificed for histological and chemical investigation. With regard to the development of epidermal disease, the results showed little difference between the reference mesocosm and the indirectly polluted mesocosm, with the exception that lymphocystis was significantly elevated in the indirectly polluted mesocosm. Although pollution may be a risk factor in the etiology of this disease, such a relationship would probably be obscured under field conditions due to variation arising from other factors. Histopathological analysis of the livers revealed in total four cases of hepatocellular adenoma (1.5% of sampled population) in fish from the polluted mesocosms, the first occurring after 2.5 years of exposure in fish from the indirectly polluted mesocosm. Furthermore, several other liver lesions, including foci of cellular alteration and hydropic vacuolated lesions, developed during the course of the experiment before tumor formation was apparent. Prevalences of these conditions were very much lower in the reference mesocosm than in the two polluted mesocosms. Densities of melanomacrophage centers in the liver showed a similar trend. The findings clearly indicate that long-term exposure to chemically contaminated dredged spoil can induce liver neoplasia and other liver lesions in flounder at contaminant levels comparable to those found in the natural environment.

Increased susceptibility to ultraviolet-B and carcinogens of mice lacking the DNA excision repair gene XPA.

Xeroderma pigmentosum patients with a defect in the nucleotide-excision repair gene XPA are characterized by, for example, a > 1,000-fold higher risk of developing sunlight-induced skin cancer. Nucleotide-excision repair (NER) is involved in the removal of a wide spectrum of DNA lesions. The XPA protein functions in a pre-incision step, the recognition of DNA damage. To permit the functional analysis of the XPA gene in vivo, we have generated XPA-deficient mice by gene targeting in embryonic stem cells. The XPA-/-mice appear normal, at least until the age of 13 months. XPA-/-mice are highly susceptible to ultraviolet (UV)-B-induced skin and eye tumours and to 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin tumours. We conclude that the XPA-deficient mice strongly mimic the phenotype of humans with xeroderma pigmentosum.

Frequent p53 alterations but low incidence of ras mutations in UV-B-induced skin tumors of hairless mice.

We have investigated UV-B-induced skin tumors of hairless SKH-HRA mice for alterations in the p53 gene and for mutations in either of the three ras genes. Out of 32 tumors screened, only one contained a ras mutation, i.e. in codon 12 of the K-ras gene. Alterations in the p53 gene were much more abundant, as illustrated immunohistochemically by the accumulation of p53 protein in 75% of the tumor sections examined. Immunoreactivity was observed primarily in the proliferative cell compartment, but no clear correlation between p53 staining in tumor cells and histological parameters for malignancy was observed. Subsequent sequence analysis showed that point mutations in the p53 gene are detectable in 30% (nine out of 30) of the skin tumors examined. The majority of the mutations are located in codons 267 and 272, most likely originating from UV-B-induced photo-adducts at dipyrimidine sites in the non-transcribed strand. Codon 272 corresponds to the human codon 278, which is also a hotspot for p53 mutations in human non-melanoma skin cancers. Codon 267 matches the human codon 273, which does not contain a dipyrimidine site, but represents a CpG hotspot for p53 mutations in internal malignancies. Our results demonstrate that this hairless mouse model for UV-induced skin cancer corresponds closely to human non-melanoma skin cancers with respect to mutations in the p53 gene.

Fish as biomarkers in immunotoxicology.

This paper presents an overview on the state of the art in the development and application of biomarkers for immunotoxicology in fish. There are several reasons for developing this field: many fish diseases are related to environmental quality, various environmental pollutants have immunotoxic potential and many fish diseases have an immunological component. As in immunotoxicology in general, this aspect, in fish, has received ample attention in the recent past. Much benefit has been obtained from progress in related fields of science, such as fish immunology and rodent immunotoxicology. Meanwhile there is a broad spectrum of potential biomarkers for immunotoxicology in fish, from which macrophage parameters seem to be most widely used. The application of others, such as lymphoid cell parameters is still limited, probably due to practical problems such as lack of experience with conduct, validation and interpretation. Specific problems include the paucity of background data in the case of epidemiological field studies and the important role of other (non-chemical) stress factors in the immune response, and hence the lack of specificity of potential biomarkers.

Toxicological pathology in laboratory fish: an evaluation with two species and various environmental contaminants.

: This paper presents an overview of the studies carried out in small laboratory fish species to investigate the usefulness of histopathology as a tool in aquatic toxicology. The studies were performed with medaka (Oryzias latipes) and guppy (Poecilia reticulata) that were exposed to a variety of environmental contaminants for one and three months. Besides the measurement of routine toxicological parameters, total body histopathology was carried out. The data are summarized and evaluated in view of their contribution to the knowledge of toxicology, the specific responsiveness of animals and tissues, and the application in ecotoxicology. It is concluded that histopathology of (small) fish exposed to environmental contaminants may provide useful information as to target organs and mechanism of action: moreover, this technique can be more sensitive than routine parameters.

Histopathological effects in Poecilia reticulata (guppy) exposed to methyl mercury chloride.

To evaluate the usefulness of histopathology in aquatic toxicity testing, studies were carried out on the small freshwater fish Poecilia reticulata (guppy) following aqueous methyl mercury chloride exposure. Fish were exposed to concentrations of 0, 1.0, 1.8, 3.2, 5.6, or 10 micrograms/L for 1 and 3 months. Histopathological changes included the occurrence of multiple granulomas in various tissues, in particular, the integument and orbit. These changes were accompanied by hyperplasia of monocytopoietic interrenal tissue, and hepatocellular change which was confirmed by morphometry. The latter findings were probably a result of monocyte "consumption" by granulomas, and hepatic synthesis of ("stress") proteins, respectively. The bile duct and, focally, the proximal intestine, showed hyperplasia of the epithelium. In the testis of sexually mature fish (3-month study), degeneration and necrosis of sperm occurred, with severe cases exhibiting Sertoli cell hypertrophy, interstitial inflammation, and absence of mature sperm. Epidermal mucous cells disappeared in the highest concentration used, and, after 3 months, clusters of undifferentiated basophilic cells were seen in the gas gland, which occasionally were suggestive of malignant growth. The changes in the kidney tubules were characterized by degeneration and necrosis of single cells which also showed mitotic figures. This is considered a result of the mitosis-disturbing activity of methyl mercury (MeHg). It is concluded that MeHg has effects on various target organs in guppies with the occurrence of granulomas as the most sensitive indicator, yielding a no-observed-effect concentration (NOEC) of 1.0 micrograms/L. In contrast to mammalian species, no morphologic evidence for neurotoxicity was found.

Histopathological effects of environmental pollutants beta-HCH and methyl mercury on reproductive organs in freshwater fish.

From various environmental pollutants studied so far, specific effects on the reproductive system of small fish species Poecilia reticulata (guppy) and Oryzias latipes (medaka) were noted in the case of beta-hexachlorocyclohexane (induction of vitellogenesis and hermaphroditism, both indicative of estrogenic activity; 32 micrograms/l) and methyl mercury (impaired spermatogenesis; 1.8 micrograms/l). The latter effect was attributed to a disturbance of mitosis.