Macrophages are involved in hexachlorobenzene-induced adverse immune effects.

Hexachlorobenzene (HCB) is a persistent environmental pollutant that causes adverse immune effects in man and rat. The Brown Norway (BN) rat is very susceptible to HCB-induced immunopathology and oral exposure causes inflammatory skin and lung lesions, splenomegaly, lymph node (LN) enlargement, and increased serum levels of IgE and anti-ssDNA IgM. T cells play an important role but do not account for all adverse effects induced by HCB. Macrophages are probably also important and the relationship between macrophages and T cells was further investigated. To eliminate macrophages clodronate-liposomes were used. Furthermore, a kinetic study was performed to obtain insight in the early phase of the HCB-induced immune response. Also, experiments were performed to detect specific memory T cells. Therefore, an adoptive transfer study was performed. Our results indicate that macrophages are indeed involved in HCB-induced skin lesions, lung eosinophilia, and elevation of IgM against ssDNA. Kinetics showed that both skin and lung lesions appeared early after exposure. Moreover, immune effects could not be adaptively transferred. Thus, both macrophages and T cells are involved in HCB-induced immune effects but HCB exposure does not lead to specific T cell sensitization. Presumably, HCB exposure induces macrophage activation, thereby generating adjuvant signals that polyclonally stimulate T cells. Together, these events may lead to the observed immunopathology in BN rats.

2-AAF-induced tumor development in nucleotide excision repair-deficient mice is associated with a defect in global genome repair but not with transcription coupled repair.

The nucleotide excision repair (NER) pathway comprises two sub-pathways, transcription coupled repair (TCR) and global genome repair (GGR). To establish the importance of these separate sub-pathways in tumor suppression, we exposed mice deficient for either TCR (Csb), GGR (Xpc) or both (Xpa) to 300 ppm 2-acetylaminofluorene (in feed, ad libitum) in a unique comparative exposure experiment. We found that cancer proneness was directly linked to a defect in the GGR pathway of NER as both Xpa and Xpc mice developed significantly more liver tumors upon 2-AAF exposure than wild type or Csb mice. In contrast, a defect in TCR appeared to act tumor suppressive, leading to a lower hepatocellular tumor response in Xpa mice (tumor incidence of 25%) as compared to Xpc mice (53% tumor-bearing mice). The link between deficient GGR and tumor proneness was most pronounced in the liver, but this phenomenon was also found in the urinary bladder. As tumor induction by 2-AAF appeared almost exclusively dependent on a defect in GGR, we examined whether gene mutation induction in the non-transcribed lacZ locus could reliably predict tumor risk. Interestingly, however, short-term 2-AAF exposure induced lacZ mutant levels in Csb mice almost as high as those found in Xpa or Xpc mice. This indicates that lacZ mutant frequencies are not correlated with a specific DNA repair defect and eventual tumor outcome, at least not in the experimental design presented here.

Comparative toxicological pathology in mammals and fish: some examples with endocrine disrupters.

Toxicologic pathology is a classical discipline in the toxicology arena, and despite various emerging techniques, still is a major cornerstone in the process of hazard identification and risk characterization. Most knowledge derives from laboratory animal studies and, to a lesser extent, human data. Currently interest is growing in applying toxicological pathology for lower animals, in particular fish as being the most developed aquatic genus. This is triggered by the interest in so-called endocrine disrupting chemicals (endocrine disrupters, EDCs), xenobiotics that interfere with the endocrine system and thus may affect reproduction and/or development, and for which pathology is an essential technique in general in vivo studies. As the aquatic ecosystem is a major recipient of pollutants, fish constitute an important potential target and can be used as a research and bio-monitoring tool. For this goal knowledge of the pathological responses of fish to EDCs is essential and therefore we have studied the responses of laboratory fish to a set of reference endocrine modulating chemicals. In this paper, such effects are compared with known response patterns in mammals, thereby accounting for the specific aspects of anatomy and physiology in fish.

p53 heterozygosity results in an increased 2-acetylaminofluorene-induced urinary bladder but not liver tumor response in DNA repair-deficient Xpa mice.

Both nucleotide excision repair (NER) and the p53 tumor suppressor protein play crucial roles in the prevention of cells becoming cancerous. This is clearly demonstrated by the fact that NER-deficient xeroderma pigmentosum patients and Li-Fraumeni patients who carry a germ-line p53 mutation are highly tumor prone. The NER-deficient Xpa and the p53(+/-) mouse models clearly mimic their human counterparts, because they are both tumor prone as well. The aim of the study presented here was to analyze the relative contribution of these two pathways in tumor suppression and to analyze a possible link between NER and p53 activation in vivo. For this, we exposed Xpa, p53(+/-), and Xpa/p53(+/-) mice to 2-acetylaminofluorene (2-AAF). We show that 2-AAF-induced urinary bladder tumor suppression is dependent on p53 status, because p53(+/-) mice were highly tumor prone. Xpa/p53(+/-) mice were even more tumor prone, whereas no increased tumor response was found in Xpa mice. Short-term assays revealed a decreased apoptotic response in Xpa/p53(+/-) mice, pointing in vivo toward a link between NER and p53-mediated apoptosis. In contrast, liver tumor response was primarily dependent on appropriate DNA repair, because Xpa-deficient mice were liver tumor prone. p53 heterozygosity had no influence on liver tumor incidences, in line with the results obtained from the short-term 2-AAF studies revealing no altered cellular response in p53(+/-) or Xpa/p53(+/-) mice. Interestingly, however, mice completely deficient in both NER and p53 (Xpa/p53(-/-) mice) showed a dramatic increase of hepatocellular proliferation accompanied by lacZ reporter gene mutations.

Hexachlorobenzene-induced Immunopathology in Brown Norway rats is partly mediated by T cells.

Hexachlorobenzene (HCB) is a persistent environmental pollutant with (auto)immune effects in humans and rats. The Brown Norway (BN) rat is very susceptible to HCB-induced immunopathology, and oral exposure causes inflammatory skin and lung lesions, splenomegaly, lymph node (LN) enlargement, and increased serum levels of IgE and anti-ssDNA IgM. The role of T cells in HCB-induced immunopathology is unclear and to elucidate this Cyclosporin A (CsA) was used. BN rats were exposed to either a control diet or a diet supplemented with 450 mg/kg HCB for 21 days. CsA treatment started 2 days prior to HCB exposure and rats were injected daily with 20 mg/kg body weight CsA. Treatment with CsA prevented the HCB-induced immunopathology significantly. The onset of skin lesions was delayed and the severity was also strongly decreased. Furthermore, CsA prevented the HCB-induced increase in spleen weight partly and the increase in auricular LN weight completely. The increase in serum IgE and IgM against ssDNA levels was prevented completely. Macrophage infiltrations into the spleen and lung still occurred but infiltrations of eosinophilic granulocytes into the lung were prevented. Restimulation of spleen cells with the T-cell mitogen ConA and the macrophage activator LPS clearly showed that CsA inhibited T-cell activation, but not macrophage activation. Together, our results show that both T cells and macrophages play a prominent role in HCB-induced immunopathology.

Persistent hematologic and immunologic disturbances in 8-year-old Dutch children associated with perinatal dioxin exposure.

Perinatal exposure to Dutch "background" dioxin levels in 1990 was high, but comparable with that of other industrialized Western European countries. Exposure during the sensitive perinatal period may cause permanent disturbances. Therefore, we assessed the health status and various hematologic and immunologic parameters among our longitudinal cohort. A medical history was taken and venipuncture performed in a longitudinal cohort of 27 healthy 8-year-old children who had documented perinatal dioxin exposure. Linear regression revealed a decrease in allergy in relation to prenatal (p = 0.02) and postnatal (p = 0.03) dioxin exposure. Increases in CD4+ T-helper cells (p = 0.006) and in CD45RA+ cells (p = 0.02) were seen in relation to postnatal exposure. A persistently decreased platelet count (p = 0.04) and increased thrombopoietin concentration (p = 0.03) were seen in relation to postnatal exposure. This follow-up has shown a decrease in allergy, persistently decreased thrombocytes, increased thrombopoietin, and increased CD4+ T-helper and increased CD45RA+ cell counts. This study provides indications of effects at the stem cell level of perinatal dioxin exposure, persisting until minimally 8 years after birth.

Combined oral benzo[a]pyrene and inhalatory ozone exposure have no effect on lung tumor development in DNA repair-deficient Xpa mice.

There is considerable concern about an enhanced risk of lung tumor development upon exposure of humans to polycyclic aromatic hydrocarbons (PAHs), like benzo[a] pyrene (B[a]P), in combination with induced lung cell proliferation by toxic agents like ozone. We studied this issue in wild-type (WT) C57BL/6 mice, the cancer prone nucleotide excision repair-deficient Xeroderma pigmentosum complementation group A mice (Xpa-/-) and the even more sensitive Xpa-/-/p53+/- mice. The mice were treated with B[a]P through the diet at a dose of 75 p.p.m., in combination with intermittent ozone exposures (0.8 p.p.m.). First, a dose-range finding study with WT and Xpa-/- mice was conducted to determine the optimal ozone concentration giving high cell proliferation and low toxic side effects. We show by BrdU incorporation that cell proliferation in the lung was induced by ozone, with an optimal concentration of 0.8 p.p.m., which was subsequently used in the (sub)chronic studies. In the subchronic study, in which lacZ mutant frequency and BPDE-DNA adduct formation were measured, the mice were treated for 13 weeks with B[a]P and/or ozone, whereas in the chronic study this treatment protocol was followed by a 6 month period on control feed and filtered air. As expected, oral B[a]P exposure appeared to be highly carcinogenic to Xpa-/- and Xpa-/-/p53+/- mice and to a lesser extent to WT mice. A high incidence of forestomach tumors and some tumors of the esophagus were found. In the lung, a clear genotoxic effect of B[a]P was found as shown by the presence of BPDE-DNA adducts. However, these DNA adducts in combination with induction of cell proliferation did not result in increased lacZ mutations, nor in lung tumor formation not even in the highly sensitive Xpa-/- and Xpa-/-/p53+/- mice. The implication of these findings for tumor risk assessment will be discussed.