Suberoylanilide hydroxamic acid affects γH2AX expression in osteosarcoma, atypical teratoid rhabdoid tumor and normal tissue cell lines after irradiation.

PURPOSE: Osteosarcoma and atypical teratoid rhabdoid tumors are tumor entities with varying response to common standard therapy protocols. Histone acetylation affects chromatin structure and gene expression which are considered to influence radiation sensitivity. The aim of this study was to investigate the effect of the combination therapy with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) and irradiation on atypical teratoid rhabdoid tumors and osteosarcoma compared to normal tissue cell lines. METHODS: Clonogenic assay was used to determine cell survival. DNA double-strand breaks (DSB) were examined by pulsed-field electrophoresis (PFGE) as well as by γH2AX immunostaining involving flow cytometry, fluorescence microscopy, and immunoblot analysis. RESULTS: SAHA lead to an increased radiosensitivity in tumor but not in normal tissue cell lines. γH2AX expression as an indicator for DSB was significantly increased when SAHA was applied 24 h before irradiation to the sarcoma cell cultures. In contrast, γH2AX expression in the normal tissue cell lines was significantly reduced when irradiation was combined with SAHA. Analysis of initial DNA fragmentation and fragment rejoining by PFGE, however, did not reveal differences in response to the SAHA pretreatment for either cell type. CONCLUSION: SAHA increases radiosensitivity in tumor but not normal tissue cell lines. The increased H2AX phosphorylation status of the SAHA-treated tumor cells post irradiation likely reflects its delayed dephosphorylation within the DNA damage signal decay rather than chromatin acetylation-dependent differences in the overall efficacy of DSB induction and rejoining. The results support the hypothesis that combining SAHA with irradiation may provide a promising strategy in the treatment of solid tumors.

Genetic polymorphisms in DNA repair and damage response genes and late normal tissue complications of radiotherapy for breast cancer.

Breast-conserving surgery followed by radiotherapy is effective in reducing recurrence; however, telangiectasia and fibrosis can occur as late skin side effects. As radiotherapy acts through producing DNA damage, we investigated whether genetic variation in DNA repair and damage response confers increased susceptibility to develop late normal skin complications. Breast cancer patients who received radiotherapy after breast-conserving surgery were examined for late complications of radiotherapy after a median follow-up time of 51 months. Polymorphisms in genes involved in DNA repair (APEX1, XRCC1, XRCC2, XRCC3, XPD) and damage response (TP53, P21) were determined. Associations between telangiectasia and genotypes were assessed among 409 patients, using multivariate logistic regression. A total of 131 patients presented with telangiectasia and 28 patients with fibrosis. Patients with variant TP53 genotypes either for the Arg72Pro or the PIN3 polymorphism were at increased risk of telangiectasia. The odds ratios (OR) were 1.66 (95% confidence interval (CI): 1.02-2.72) for 72Pro carriers and 1.95 (95% CI: 1.13-3.35) for PIN3 A2 allele carriers compared with non-carriers. The TP53 haplotype containing both variant alleles was associated with almost a two-fold increase in risk (OR 1.97, 95% CI: 1.11-3.52) for telangiectasia. Variants in the TP53 gene may therefore modify the risk of late skin toxicity after radiotherapy.

[Epigenetic aspects in carcinomas of the head and neck]. / Epigenetische Aspekte bei Karzinomen der Kopf-Hals-Region.

For years, head and neck squamous cell carcinomas (HNSCC) have been among the leading cancers worldwide. Despite considerable efforts, the 5-year survival rate for HNSCC has not changed significantly. To improve this situation, it is necessary to understand the fundamental biological processes leading to the disease and its progression. In addition to known genetic changes in HNSCC, molecular cytogenetic investigations have identified chromosomal regions of gains and losses, but many of the responsible candidate genes have yet to be identified. Furthermore, recent results indicate the importance of epigenetic modifications in HNSCC, such as DNA methylation. Several genes, including the tumor suppressor CDKN2A and other candidates such as DAPK1, MGMT, TIMP3, TCF21, and C/EBPalpha, have been found to harbor hypermethylated regulatory sequences that lead to reduced expression or gene silencing. Hypermethylation in such genes could be used not only as biomarkers for the early detection of HNSCC but also to improve prevention strategies and therapy outcomes.

DNA damaging capability of hematoporphyrin towards DNAs of various accessibilities.

In this work we wanted to verify that photoactivation of DNA-non-binding porphyrin derivative hematoporphyrin IX (Hp) is able to induce damages in DNAs of various accessibilities such as B-conformation and superhelical isolated DNA, nucleoprotein complex and intracellular DNAs. It was found that photodynamic reaction of Hp results significant changes in thermal stability of isolated T7 DNA and induces single strand breaks in supercoiled Bluescript plasmid isolated from Escherichia coli cells. As optical melting measurements revealed, the irradiation of photosensitized T7 nucleoprotein complex leads to a destabilization of the protein capsid. The photodynamic reaction affected both the protein structure and DNA-protein interaction, however, the parameters corresponding to the DNA denaturation are not influenced. The accumulation of Hp in HeLa cells was followed by laser scanning confocal microscopy. The picture received is typical for lipophilic dyes. When Hp loaded cells were irradiated, a reduction of viability could be observed in a concentration and a light dose dependent manner; 12microM porphyrin induced almost complete cell killing after 30min irradiation. After similar treatment, alkaline agarose gel electrophoresis of isolated nuclear DNA did not show the presence of single strand breaks. The alkaline comet assay also failed to demonstrate any DNA damage in HeLa cells. We also considered the possibility of the generation of damages in intracellular SV40 DNA. According to the electropherograms there was no difference between the patterns of DNAs from treated and control samples.

Epigenetics in radiation-induced fibrosis.

Radiotherapy is a major cancer treatment option but dose-limiting side effects such as late-onset fibrosis in the irradiated tissue severely impair quality of life in cancer survivors. Efforts to explain radiation-induced fibrosis, for example, by genetic variation remained largely inconclusive. Recently published molecular analyses on radiation response and fibrogenesis showed a prominent role of epigenetic gene regulation. This review summarizes the current knowledge on epigenetic modifications in fibrotic disease and radiation response, and it points out the important role for epigenetic mechanisms such as DNA methylation, microRNAs and histone modifications in the development of this disease. The synopsis illustrates the complexity of radiation-induced fibrosis and reveals the need for investigations to further unravel its molecular mechanisms. Importantly, epigenetic changes are long-term determinants of gene expression and can therefore support those mechanisms that induce and perpetuate fibrogenesis even in the absence of the initial damaging stimulus. Future work must comprise the interconnection of acute radiation response and long-lasting epigenetic effects in order to assess their role in late-onset radiation fibrosis. An improved understanding of the underlying biology is fundamental to better comprehend the origin of this disease and to improve both preventive and therapeutic strategies.

Glutathione-S-transferase M1, M3, T1 and P1 polymorphisms and susceptibility to non-small-cell lung cancer subtypes and hamartomas.

Polymorphic glutathione-S-transferase (GST) genes causing variations in enzyme activity may influence individual susceptibility to lung cancer. In this case-control study (consisting of 389 Caucasian lung cancer patients, including 151 adenocarcinomas (ACs) and 172 squamous cell carcinomas (SCCs), and 353 hospital control subjects without malignant disease, genotype frequencies for GSTM1, GSTM3, GSTP1 and GSTT1 were determined by polymerase chain reaction (PCR)/ restriction fragment length polymorphism (RFLP)-based methods. While adjusted odds ratios (ORs) indicated no significantly increased risk for lung cancer overall due to any single GST genotype, the risk alleles for GSTM1, GSTM3 and GSTP1 conferring reduced enzyme activity were present at higher frequency in SCC than in AC patients. This is consistent with a reduced detoxification of carcinogenic polycyclic aromatic hydrocarbons (PAHs) from cigarette smoke that are more important for the development of SCC than for AC. An explorative data analysis also identified statistically significantly increased ORs for the combinations GSTT1 non-null and GSTP1 GG or AG for lung cancer overall (OR 2.23, CI 1.11-4.45), and for SCC (OR 2.69, CI 1.03-6.99). For lung cancer overall, and especially among SCC patients, the GSTT1 null genotype was underrepresented (SCC 11.2% v. control subjects 19%, P = 0.026, OR 0.57, CI 0.30-1.06). Additionally, in 28 patients with hamartomas, the GSTT1 null genotype was also protective (P = 0.013), while GSTP1 variant allele carriers were overrepresented (OR 2.48, CI 1.06-6.51). In conclusion, GST genotypes may act differently, either by detoxifying harmful tobacco carcinogens and/or by eliminating lung cancer chemopreventive agents. The latter role for GSTT1 would explain the observed lower risk of SCC and hamartoma associated with GSTT1 null. Further confirmatory studies are required.

Relevance of N-acetyltransferase 1 and 2 (NAT1, NAT2) genetic polymorphisms in non-small cell lung cancer susceptibility.

The highly polymorphic N-acetyltransferases (NAT1 and NAT2) are involved in both activation and inactivation reactions of numerous carcinogens, such as tobacco derived aromatic amines. The potential effect of the NAT genotypes in individual susceptibility to lung cancer was examined in a hospital based case-control study consisting of 392 Caucasian lung cancer patients [152 adenocarcinomas, 173 squamous cell carcinomas (SCC) and 67 other primary lung tumours] and 351 controls. In addition to the wild-type allele NAT1*4, seven variant NAT1 alleles (NAT1*3, *10, *11, *14, *15, *17 and *22) were analysed. A new method based on the LightCycler (Roche Diagnostics Inc.) technology was applied for the detection of the polymorphic NAT1 sites at nt 1088 and nt 1095. The NAT2 polymorphic sites at nt 481, 590, 803 and 857 were detected by polymerase chain reaction-restriction fragment length polymorphism or LightCycler. Multivariate logistic regression analyses were performed taking into account levels of smoking, age, gender and occupational exposure. An increased risk for adenocarcinoma among the NAT1 putative fast acetylators [odds ratio (OR) 1.92 (1.16-3.16)] was found but could not be detected for SCC or the total case group. NAT2 genotypes alone appeared not to modify individual lung cancer risk, however, individuals with combined NAT1 fast and NAT2 slow genotype had significantly elevated adenocarcinoma risk [OR 2.22 (1.03-4.81)] compared to persons with other genotype combinations. These data clearly show the importance of separating different histological lung tumour subtypes in studies on genetic susceptibility factors and implicate the NAT1*10 allele as a risk factor for adenocarcinoma.

Quantitative assessment of bleomycin-induced poly(ADP-ribosyl)ation in human lymphocytes by immunofluorescence and image analysis.

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme that is catalytically activated by DNA strand interruptions. It catalyses the covalent modification of proteins with ADP-ribose polymers, using NAD(+) as precursor. Here, we have studied the DNA damage-induced formation of poly(ADP-ribose) in intact human peripheral blood lymphocytes (PBL) by in-situ immunofluorescence detection. The response of PBL to bleomycin (BLM), which is known to induce DNA single and double strand breaks, was investigated with regard to polymer formation. For this purpose, a quantitative approach was developed to assess more accurately the immunostaining of polymer formation by computerised image analysis. As an application of this new method, we have determined the polymer formation following BLM treatment in quiescent human PBL versus mitogen activated cells. Quiescent human PBL showed a similar basal immunostaining for the polymer compared to phytohemagglutinin (PHA)-activated cells, expressed as relative mean pixel intensity (RMPI) (1.3+/-0.8 and 2.2+/-0.9, respectively; P<0.3). After BLM treatment, there was a clear-cut enhancement of polymer immunostaining, with PHA-activated cells showing significantly higher RMPI than non-activated cells (9.2+/-1.4 and 4.2+/-1.0, respectively; P<0.005). As expected, in the presence of the ADP-ribosylation inhibitor 3-aminobenzamide (3-AB), the RMPI of immunostained polymer was decreased in both quiescent and PHA-activated PBL to 1.2+/-0.7 and 1.5+/-0.9, respectively. Our findings reveal (i) that mitogen-stimulated, intact lymphocytes show enhanced polymer formation following BLM treatment, and (ii) that our new quantitative immunofluorescence assay coupled with computerised image analysis is reliable and sensitive enough to detect changes in polymer formation rate.

Sugar-linked dithiocarbamates as modulators of metabolic and genotoxic properties of N-nitroso compounds.

A series of putative anticarcinogenic and antimutagenic compounds was synthesized on the basis of tetraethylthiuram disulfide (disulfiram) and its metabolite, diethyldithiocarbamate (DDTC). Diallyldithiocarbamate was synthesized in order to combine the anticarcinogenic properties of diallyl sulfide, a known inhibitor of chemical carcinogenesis from Allium species, and those of DDTC. Several sugar-linked dithiocarbamates (SDTCs) were prepared using glucose, cellobiose, and lactose as glycosyl donors and DDTC and diallyldithiocarbamate as acceptors. All the S--glycoside bonds of SDTCs were very stable under physiological conditions in vitro. At low nitrosamine concentrations, glucose-DDTC inhibited microsomal nitrosamine dealkylases in vitro. In vivo these enzymes were also inhibited 4 h after i.p. administration of glucose-DDTC or lactose-DDTC to rats (1.7 mmol/kg); after 24 h, the values had returned to control levels. Glucose-DDTC induced the activity of glutathione-related enzymes. Concomitant treatment of rats with glucose-DDTC and N-nitrosodiethylamine (NDEA) led to a depression of the oxidative metabolism of [14C]NDEA to 14CO2 but increased the elimination of unchanged [14C]NDEA in the urine. Furthermore, glucose-DDTC totally inhibited the formation of DNA single-strand breaks induced by NDEA. All these effects may contribute to possible antimutagenic and anticarcinogenic actions of the dithiocarbamates investigated.

Urinary excretion of N-nitrosodimethylamine in rats after Thalamonal narcosis.

Urinary excretion of N-nitrosodimethylamine (NDMA) in Sprague--Dawley rats was investigated after oral administration and inhalation of NDMA and concomitant narcosis by Thalamonal and diethyl ether. While ether anesthesia induced a 4-fold increase in the excretion rate, there was a drastic reduction (about 20-fold) in the amount of NDMA excreted after narcosis by Thalamonal.

Lipid peroxidation status, somatic mutations and micronuclei in peripheral lymphocytes: a case observation on a possible interrelationship.

A controlled dietary study was conducted in healthy female volunteers and reported elsewhere [1]. In a subset of samples four different biomarkers were analyzed: plasma malondialdehyde (MDA) levels and urinary 8-isoprostaglandin-F(2alpha) were measured as markers for lipid peroxidation. The frequency of hprt (hypoxanthine guanine phosphoribosyl transferase) mutants and micronuclei in peripheral blood lymphocytes were analyzed as indicators of genotoxic effects. One of the ten individuals showed extremely high background levels in all of the four endpoints measured. This case observation raises the possibility that life style factors and dietary habits affect the level of DNA reactive lipid peroxidation products, which in turn increase mutagenic and cytogenetic effects. A possible association between these biomarkers, particularly in relation to dietary fat intake and antioxidant status, should now be studied in a larger trial.

Carcinogenicity of small doses of inhaled N-nitrosoacetoxymethyl-N-methylamine in Syrian golden hamsters.

Male Syrian golden hamsters inhaled 0.5-1 ppm (= 2.7-5.5 mg/m3) of N-nitroso-N-acetoxy-methyl-N-methylamine 1 h/week, for 14 weeks. The total dose per animal was calculated as 150-400 micrograms or 1-3 mg/kg. Four squamous cell carcinomas and one mucoepidermoid carcinoma of the nasal mucosa were observed. No such tumors occurred in the control group.

Quantitative analysis of the early changes of hepatocyte nuclei after treating Syrian golden hamsters with di(2-ethylhexyl)phthalate.

Although the biological action of phthalates has been widely discussed there is little information on early cellular changes indicative for toxic or carcinogenic effects. To study subtle alterations in the cell morphology, we have by means of image processing evaluated the nuclei of hamster hepatocytes after treatment with di(2-ethylhexyl)phthalate given in single i.p. doses of 30, 300, and 3000 mg/kg. The results indicate that by using specially developed methods for analysis of images of cell nuclei and chromatin structure, it is possible to recognize changes eluding detection with usual light microscopy.

Genotoxic activities of benzamidine and its N-hydroxylated metabolite benzamidoxime in Salmonella typhimurium and mammalian cells.

The genotoxic potentials of benzamidine and benzamidoxime were determined to study the toxicological relevance of the metabolic N-oxygenation (N-hydroxylation) of benzamidines to benzamidoximes. Benzamidoxime induced DNA single-strand breaks (in rat hepatocytes) and DNA amplification in SV40-transformed hamster cells. In the experiments performed, benzamidine itself was only marginally positive in the hepatocyte/DNA single-strand break assay. Since these cells possess an intact metabolization apparatus, the biological activities may be attributed to toxic and genotoxic metabolites formed by biotransformation. In the Salmonella typhimurium mutagenicity test (TA 98 and TA 100) benzamidoxime alone exhibited a low mutagenicity in the TA 98 strain in the presence of rabbit liver S-9 fractions. These results permit recognition of the metabolic N-hydroxylation of benzamidines to benzamidoximes as a process to toxication. Indirect evidence for the formation of a glucuronide of benzamidoxime has been obtained from in vitro experiments, but it could not be established that this process was a decisive factor in the genotoxicity of benzamidoxime.

Mutagenic and genotoxic activities of extracts derived from the cooked and raw edible mushroom Agaricus bisporus.

A. bisporus has been reported to be carcinogenic to mice [Toth et al. (1986) Cancer Res 38:177-180] and mutagenic in Salmonella typhimurium [Sterner et al. (1982) Mutat Res 101:269-281]. The effects of different heat treatments on the mutagenicity of raw, cooked (boiled) and fried A. bisporus extracts in the S. typhimurium test is reported. The spectrum of potential mutagenic activity of A. bisporus extracts was tested in vitro in Syrian hamster embryo cells for selective DNA amplification and in primary rat hepatocytes for DNA single-strand breaks. DNA single-strand breaks were also determined in liver cells of rats and micronuclei were measured in bone marrow cells of mice in vivo following oral application of A. bisporus extracts. It was shown that the complex A. bisporus extracts per se are not detectably mutagenic in S. typhimurium and that the previously observed increase in number of colonies per plate is probably due to a histidine artefact. No indication of genotoxicity was seen in the two in vitro assays with primary mammalian cells with two different end points. No evidence of in vivo genotoxic effects was observed in the rat liver cells. Finally, A. bisporus was not genotoxic in the micronucleus assay of mouse bone marrow cells in contrast to its previously reported carcinogenicity in mice.

Determination of DNA single strand breaks and selective DNA amplification by N-nitrodimethylamine and analogs, and estimation of the indicator cells' metabolic capacities.

N-nitrodimethylamine is metabolized oxidatively to N-nitrohydroxymethylmethylamine, which decomposes to yield formaldehyde and N-nitromethylamine. All four compounds and N-nitromethylamine were tested for their ability to induce DNA single strand breaks in hepatocytes and in SV 40-transformed Chinese hamster embryo cell lines. Only the two monoalkylnitramines were positive. They induced single strand breaks in hepatocytes, but were not effective in the other cells. Formaldehyde and N-nitrohydroxymethylmethylamine were toxic to the cells. None of the compounds tested was able to induce selective DNA amplification in the two transformed cell lines. Enzymes involved in drug metabolism were assayed in the hamster cell lines. The activity of UDP-glucuronosyltransferase and cytosolic epoxide hydrolase were not detectable. N-nitrodimethylamine demethylation was low. The content of reduced glutathione and the activities of glutathione transferase and membrane bound epoxide hydrolase were comparable to values obtained in the rat liver.

An investigation of the DNA-damaging ability of benzene and its metabolites in human lymphocytes, using the comet assay.

Benzene and five of its known metabolites--muconic acid, hydroquinone, catechol, p-benzoquinone, and benzentriol--were examined for DNA damage in human lymphocytes using the alkaline Comet assay, and conditions were optimised to determine responses. Metabolic activation (S-9 mix) was included in the assay for varying times to try to enhance effects. In addition, the effects of catalase were investigated as it is known to be present in S-9 mix reducing oxidative damage, and some benzene metabolites are known to react through oxygen radical mechanisms. Effects were also examined in cycling cells to determine whether they were more sensitive to damage then noncycling cells. Comets were measured either by eye or by image analysis. Data have been presented according to length of treatments. When Comets were measured by eye after treatment with hydrogen peroxide (H2O2), the positive control, and each compound for 0.5 hr, only H2O2 and benzenetriol induced pronounced DNA damage without metabolic activation. The effect of catechol was moderate compared with that of benzenetriol. There was a very weak effect of benzene in the absence of rat liver S-9 mix. In the presence of S-9 mix, benzene was not activated. The effect of benzenetriol was greatly reduced by the external metabolising system, but p-benzoquinone became activated to some extent. Catalase abolished the effect of benzenetriol, suggesting that H2O2 formed during autoxidation may be responsible for the DNA-damaging ability of this metabolite. The presence of catalase in S-9 mix may explain the detoxification of benzenetriol and the failure to detect consistent benzene responses. Mitogen-stimulated cycling cells were less sensitive to H2O2 and benzenetriol than unstimulated G0 lymphocytes. When comets were measured by image analysis, a 0.5-hr treatment with H2O2 and benzenetriol and catechol confirmed results analysed by eye, with S-9 mix greatly reducing responses. When treatments were increased to 1 hr in the presence and absence of S-9 mix, benzene at a 5-fold increased dose produced a significant positive response but not at the lower dose. When treatment times were increased to 2 and 4 hr, doses were also increased, and muconic acid, hydroquinone, catechol, and benzoquinone in the presence of S-9 mix showed positive time and dose-related responses, and at the highest dose of benzoquinone the morphology of the nucleus was affected. Effects tended to become more pronounced at high doses and after longer exposures, although this was not always consistent from experiment to experiment. In conclusion, benzene and all metabolites investigated gave positive responses. Where altered responses were observed, they were significantly different from the corresponding controls.

Employment of adult mammalian primary cells in toxicology: in vivo and in vitro genotoxic effects of environmentally significant N-nitrosodialkylamines in cells of the liver, lung, and kidney.

This report focuses on the use of freshly isolated primary mammalian cells from different tissues and organs of the rat for the rapid and efficient analysis of toxic and genotoxic chemicals. The cells are either treated in vitro or they are isolated from treated animals. Viability by trypan blue exclusion and DNA damage as single-strand breaks are monitored in either case. Therefore, it is possible to compare in vitro and in vivo results directly. N-nitrosamines with unique organ-specific modes in carcinogenesis were studied in vitro using hepatocytes derived from three species (rat, hamster, and pig) and in rat lung and kidney cells. The sensitive detection of all carcinogenic nitrosamines was achieved, although a pattern of cell-specific activation was not observable. The new modification of the in vivo approach allowed the sensitive detection of NDMA genotoxicity in hepatic and in extrahepatic tissues. It is important to point out that the method is an efficient tool for toxicokinetic studies with genotoxic carcinogens in vivo.

Detection of genotoxic effects in human gastric and nasal mucosa cells isolated from biopsy samples.

To assess genotoxic burdens from chemicals, it is necessary to relate observations in experimental animals to humans. The success of this extrapolation would be increased by including data on chemical activities in human tissues. Therefore, we have developed techniques to assess DNA damage in human gastric and nasal mucosa (GM, NM) cells. Biopsy samples were obtained during gastroscopy from macroscopically healthy tissue of the stomach or from healthy nasal epithelia during surgery. The specimens were incubated for 30-45 min at 37 degrees C with a digestive solution. We obtained 1.5-8 x 10(6) GM cells and 5-10 x 10(5) NM cells per donor, both with viabilities of 80-95%. The cells were incubated in vitro for 1 hr at 37 degrees C with the test compounds added in their appropriate solvents. In GM cells, we studied N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), sodium dichromate (Na2Cr2O7), nickel sulphate (NiSO4), cadmium sulphate (CdSO4), and lindane. In NM cells, lindane was investigated. Each compound was assessed for DNA damaging activity in cells of at least three different human donor samples using the microgel single cell assay. Similar studies were performed with GM and NM cells obtained from Sprague-Dawley rats. We have found human GM cells to be more sensitive to the genotoxic activity of MNNG than rat GM cells (low effective concentration [LEC] = 0.16 and 0.625 micrograms/ml for human and rat, respectively). Human cells were also more sensitive to the cytotoxic/genotoxic activity of NiSO4 (LEC = 5 and 19 mumoles/ml for human and rat, respectively). CdSO4 was genotoxic in human GM cells (LEC = 0.03-0.125 mumoles/ml), whereas no dose-related genotoxicity was observed in rat GM at concentrations up to 0.5 mumoles/ml. In contrast, approximately equal responses regarding genotoxicity and cytotoxicity were observed in rat and human GM for Na2Cr2O7 (0.25-1 mumoles/ml). Lindane, however, was genotoxic in three out of four rat GM but not in human GM cells (0.5-1 mumoles/ml), whereas it was active in both rat and human NM cells. Together with other recently published in vivo findings, our results with lindane can be interpreted according to a parallelogram approach. In view of possible human exposure situations and the sensitivities of the two target tissues from both species, the data imply that lindane will pose a health risk to humans by inhalation but not by ingestion.

Assay-specific genotoxicity of N-nitrosodibenzylamine to the rat liver in vivo.

N-Nitrosodibenzylamine (NDBzA) is mutagenic to Salmonella typhimurium and induces DNA strand breaks in isolated rat hepatocytes, yet it is reported to be non-carcinogenic to the rat. Here we report that it is inactive in both the rat and mouse bone marrow micronucleus assays and in a rat liver autoradiographic assay for unscheduled DNA synthesis. It is, however, clearly active as a micronucleus-inducing agent and mitogen in the rat liver and is capable of inducing single-strand breaks in the DNA of rat liver. The origin and implications of this curious conflict of in vivo genotoxicity data are discussed. Irrespective of that discussion, it is concluded that NDBzA is genotoxic to the rat liver in vivo.