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This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. Background and Aims: Smokeless tobacco is a heterogeneous product group with diverse composition and prevalence globally. Tobacco use during pregnancy is concerning due to the risk of adverse pregnancy outcomes and effects on child health. Nicotine may mediate several of these effects. This systematic review measured health outcomes from Swedish smokeless tobacco (snus) use during pregnancy. Method: Literature search was conducted by an information specialist in May 2022. We included human studies of snus use during pregnancy compared with no tobacco use, assessed risk of bias, conducted a meta-analysis and assessed confidence in effectestimates using Grading of Recommendations, Assessment, Development and Evaluations (GRADE). Results: We included 18 cohort studies (42 to 1 006 398 participants). Snus use during pregnancy probably (moderate confidence in risk estimates) increase the risk of neonatal apnea, adjusted odds ratio 95% confidence interval [aOR (95% CI)] 1.96 (1.30 to 2.96). Snus use during pregnancy possibly (low confidence in risk estimates) increase the risk of stillbirths aOR 1.43 (1.02 to 1.99), extremely premature births aOR 1.69 (1.17 to 2.45), moderately premature birth aOR 1.26 (1.15 to 1.38), SGA aOR 1.26 (1.09 to 1.46), reduced birth weight mean difference of 72.47 g (110.58 g to 34.35 g reduction) and oral cleft malformations aOR 1.48 (1.00 to 2.21). It is uncertain (low confidence in risk estimates, CI crossing 1) whether snus use during pregnancy affects risk of preeclampsia aOR 1.11 (0.97 to 1.28), antenatal bleeding aOR 1.15 (0.92 to 1.44) and very premature birth aOR 1.26 (0.95 to 1.66). Risk of early neonatal mortality and altered heart rate variability is uncertain, very low confidence. Snus using mothers had increased prevalence of caesarean sections, low confidence. Conclusions: This systematic review reveals that use of smokeless tobacco (snus) during pregnancy may adversely impact the developing child.

Neural stem cells (NSCs) derived from human induced pluripotent stem cells were used to investigate effects of exposure to the food contaminant acrylamide (AA) and its main metabolite glycidamide (GA) on key neurodevelopmental processes. Diet is an important source of human AA exposure for pregnant women, and AA is known to pass the placenta and the newborn may also be exposed through breast feeding after birth. The NSCs were exposed to AA and GA (1 ×10-8 - 3 ×10-3 M) under 7 days of proliferation and up to 28 days of differentiation towards a mixed culture of neurons and astrocytes. Effects on cell viability was measured using Alamar Blue™ cell viability assay, alterations in gene expression were assessed using real time PCR and RNA sequencing, and protein levels were quantified using immunocytochemistry and high content imaging. Effects of AA and GA on neurodevelopmental processes were evaluated using endpoints linked to common key events identified in the existing developmental neurotoxicity adverse outcome pathways (AOPs). Our results suggest that AA and GA at low concentrations (1 ×10-7 - 1 ×10-8 M) increased cell viability and markers of proliferation both in proliferating NSCs (7 days) and in maturing neurons after 14-28 days of differentiation. IC50 for cell death of AA and GA was 5.2 × 10-3 M and 5.8 × 10-4 M, respectively, showing about ten times higher potency for GA. Increased expression of brain derived neurotrophic factor (BDNF) concomitant with decreased synaptogenesis were observed for GA exposure (10-7 M) only at later differentiation stages, and an increased number of astrocytes (up to 3-fold) at 14 and 21 days of differentiation. Also, AA exposure gave tendency towards decreased differentiation (increased percent Nestin positive cells). After 28 days, neurite branch points and number of neurites per neuron measured by microtubule-associated protein 2 (Map2) staining decreased, while the same neurite features measured by βIII-Tubulin increased, indicating perturbation of neuronal differentiation and maturation. Developmental neurotoxicity of acrylamide and its metabolite glycidamide in a human mixed culture of neurons and astrocytes undergoing differentiation in concentrations relevant for human exposure

Air pollution is the leading environmental risk factor for disease and premature death in the world. This is mainly due to exposure to urban air particle matter (PM), in particular, fine and ultrafine combustion-derived particles (CDP) from traffic-related air pollution. PM and CDP, including particles from diesel exhaust (DEP), and cigarette smoke have been linked to various cardiovascular diseases (CVDs) including atherosclerosis, but the underlying cellular mechanisms remain unclear. Moreover, CDP typically consist of carbon cores with a complex mixture of organic chemicals such as polycyclic aromatic hydrocarbons (PAHs) adhered. The relative contribution of the carbon core and adhered soluble components to cardiovascular effects of CDP is still a matter of discussion. In the present review, we summarize evidence showing that CDP affects intracellular calcium regulation, and argue that CDP-induced impairment of normal calcium control may be a critical cellular event through which CDP exposure contributes to development or exacerbation of cardiovascular disease. Furthermore, we highlight in vitro research suggesting that adhered organic chemicals such as PAHs may be key drivers of these responses. CDP, extractable organic material from CDP (CDP-EOM), and PAHs may increase intracellular calcium levels by interacting with calcium channels like transient receptor potential (TRP) channels, and receptors such as G protein-coupled receptors (GPCR; e.g., beta-adrenergic receptors [βAR] and protease-activated receptor 2 [PAR-2]) and the aryl hydrocarbon receptor (AhR). Clarifying a possible role of calcium signaling and mechanisms involved may increase our understanding of how air pollution contributes to CVD.

Exposure to diesel exhaust particles (DEP) may contribute to endothelial dysfunction and cardiovascular disease. DEP, extractable organic material from DEP (DEP-EOM) and certain PAHs seem to trigger [Ca2+]i increase as well as inflammation via GPCRs like βARs and PAR-2. In the present study we explored the involvement of βARs and PAR-2 in effects of DEP-EOM on [Ca2+]i and expression of inflammation-associated genes in the endothelial cell-line HMEC-1. We exposed the human microvascular endothelial cell line HMEC-1 to DEP-EOM fractionated by sequential extraction with solvents of increasing polarity: n-hexane (n-Hex-EOM), dichloromethane (DCM-EOM), methanol (Methanol-EOM) and water (Water-EOM). While Methanol-EOM and Water-EOM had no marked effects, n-Hex-EOM and DCM-EOM enhanced [Ca2+]i (2–3 times baseline) and expression of inflammation-associated genes (IL-1α, IL-1β, COX-2 and CXCL8; 2–15 times baseline) in HMEC-1. The expression of βARs (60–80% of baseline) and βAR-inhibitor carazolol suppressed the increase in [Ca2+]i induced by both n-Hex- and DCM-EOM. Carazolol as well as the Ca2+-channel inhibitor SKF-96365 reduced the DCM-EOM-induced pro-inflammatory gene-expression. Overexpression of βARs increased DCM-EOM-induced [Ca2+]i responses in HEK293 cells, while βAR-overexpression suppressed [Ca2+]i responses from n-Hex-EOM. Furthermore, the PAR-2-inhibitor ENMD-1068 attenuated [Ca2+]i responses to DCM-EOM, but not n-Hex-EOM in HMEC-1. The results suggest that βAR and PAR-2 are partially involved in effects of complex mixtures of chemicals extracted from DEP on calcium signalling and inflammation-associated genes in the HMEC-1 endothelial cell-line.

Combustion-derived particles (CDPs), in particular from traffic, are regarded as a central contributor for adverse health effects linked to air pollution. Recently, also biomass burning has been recognized as an important source for CDPs. Here, the effects of CDPs (PM10) originating from burning of pellet, charcoal and wood on key processes associated to lung carcinogenesis were explored. Human bronchial epithelial cells (HBEC3-KT) were exposed to 2.5 μg/cm2 of CDPs for 24 h and biological effects were examined in terms of cytotoxicity, inflammation, epithelial to mesenchymal transition (EMT)-related effects, DNA damage and genotoxicity. Reduced cell migration, inflammation and modulation of various PM-associated genes were observed mainly after exposure to wood and pellet. In contrast, only particles from pellet burning induced alteration in cell proliferation and DNA damage, which resulted in cell cycle alterations. Charcoal instead, appeared in general less effective in inducing pro-carcinogenic effects. These results illustrate differences in the toxicological profile due to the CDPs source. The different chemical compounds adsorbed on CDPs seemed to be central for particle properties, leading to an activation of various cellular signaling pathways involved in early steps of cancer progression.

Tobacco smoking and use of snus (smokeless tobacco) are associated with adverse effects on pregnancy and neonatal outcomes. Nicotine is considered a key toxicant involved in effects caused by both smoking and snus, while pyrolysis products including polycyclic aromatic hydrocarbons (PAHs) in cigarette smoke represents the constituents most unequally divided between these two groups of tobacco products. The aim of this review was: i) to compare the impact, in terms of relative effect estimates, of cigarette smoking and use of Swedish snus on pregnancy outcomes using similar non-tobacco user controls, and ii) to examine whether exposure to PAHs from smoking could explain possible differences in impact on pregnancy outcomes. We systematically searched MEDLINE, Embase, PsycInfo, Web of Science and the Cochrane Database of Systematic Reviews up to October 2021 and identified studies reporting risks for adverse pregnancy and neonatal outcomes associated with snus use and with smoking relative to pregnant women with no use of tobacco. Both snus use and smoking were associated with increased risk of stillbirth, preterm birth, and oral cleft malformation, with comparable point estimates. These effects were likely due to comparable nicotine exposure. We also found striking differences. While both smoking and snus increased the risk of having small for gestational age (SGA) infants, risk from maternal smoking was markedly higher as was the reduction in birthweight. In contrast, the risk of preeclampsia (PE) was markedly lower in smokers than in controls, while snus use was associated with a slightly increased risk. We suggest that PAHs acting via AhR may explain the stronger effects of tobacco smoking on SGA and also to the apparent protective effect of cigarette smoking on PE. Possible mechanisms involved include: i) disrupted endocrine control of fetal development as well as placental development and function, and ii) stress adaption and immune suppression in placenta and mother.

Different transition metals have been shown to induce inflammatory responses in lung. We have compared eight different metal ions with regard to cytokine responses, cytotoxicity and signalling mechanisms in a human lung epithelial cell model (BEAS-2B). Among the metal ions tested, there were large differences with respect to pro-inflammatory potential. Exposure to Cd(2+), Zn(2+) and As(3+) induced CXCL8 and IL-6 release at concentrations below 100μM, and Mn(2+) and Ni(2+) at concentrations above 200μM. In contrast, VO4(3-), Cu(2+) and Fe(2+) did not induce any significant increase of these cytokines. An expression array of 20 inflammatory relevant genes also showed a marked up-regulation of CXCL10, IL-10, IL-13 and CSF2 by one or more of the metal ions. The most potent metals, Cd(2+), Zn(2+) and As(3+) induced highest levels of oxidative activity, and ROS appeared to be central in their CXCL8 and IL-6 responses. Activation of the MAPK p38 seemed to be a critical mediator. However, the NF-κB pathway appeared predominately to be involved only in Zn(2+)- and As(3+)-induced CXCL8 and IL-6 responses. Thus, the most potent metals Cd(2+), Zn(2+) and As(3+) seemed to induce a similar pattern for the cytokine responses, and with some exceptions, via similar signalling mechanisms.

The mycotoxin alternariol (AOH) is produced by Alternaria fungi. It occurs naturally in foodstuffs and is frequently found as contaminant in fruit and grain products. Most information regarding AOH toxicity and the potential mechanisms involved comes from in vitro studies, as only very limited in vivo studies have been performed. AOH forms reactive oxygen species (ROS) and interacts with DNA topoisomerase, thereby generating both single (SSB)- and double-strand DNA beaks (DSB). This triggers various DNA damage response pathways. AOH causes a marked reduction in proliferation in mammalian cells due to cell cycle arrest often in the G2 /M-phase. After an additional inhibition of cytokinesis, cells with abnormal nuclei as well as polyploidy are reported. In macrophages, AOH may increase autophagic activity and induce senescence. Furthermore, AOH is found to change the morphology and phenotype of various human macrophage cell models. Studies so far indicate that the AOH-induced effects are primarily a result of DSB via its effects on topoisomerase activity. Thus, most probably there will be a threshold for the AOH-induced effects, typically seen in the 5-10 μM range. These in vitro mechanistic studies further support the in vivo studies suggesting low acute toxicity. However, a decreased immune response to infections and/or a disturbed balance of the adaptive immune system when exposed together with other mycotoxins cannot be excluded. This hypothesis needs to be further explored with proper in vivo studies.

Diesel combustion and solid biomass burning are the major sources of ultrafine particles (UFP) in urbanized areas. Cardiovascular and pulmonary diseases, including lung cancer, are possible outcomes of combustion particles exposure, but differences in particles properties seem to influence their biological effects. Here the physico-chemical properties and biological effects of diesel and biomass particles, produced under controlled laboratory conditions, have been characterized. Diesel UFP were sampled from a Euro 4 light duty vehicle without DPF fuelled by commercial diesel and run over a chassis dyno. Biomass UFP were collected from a modern automatic 25 kW boiler propelled by prime quality spruce pellet. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images of both diesel and biomass samples showed aggregates of soot particles, but in biomass samples ash particles were also present. Chemical characterization showed that metals and PAHs total content was higher in diesel samples compared to biomass ones. Human bronchial epithelial (HBEC3) cells were exposed to particles for up to 2 weeks. Changes in the expression of genes involved in xenobiotic metabolism were observed after exposure to both UFP already after 24 h. However, only diesel particles modulated the expression of genes involved in inflammation, oxidative stress and epithelial-to-mesenchymal transition (EMT), increased the release of inflammatory mediators and caused phenotypical alterations, mostly after two weeks of exposure. These results show that diesel UFP affected cellular processes involved in lung and cardiovascular diseases and cancer. Biomass particles exerted low biological activity compared to diesel UFP. This evidence emphasizes that the study of different emission sources contribution to ambient PM toxicity may have a fundamental role in the development of more effective strategies for air quality improvement.

Adsorbed soluble organics seem to be the main drivers of inflammatory responses induced by diesel exhaust particles (DEP). The specific compounds contributing to this process and the cellular mechanisms behind DEP-induced inflammation are not well known. We have assessed pro-inflammatory effects of DEP and various soluble DEP fractions, in human bronchial epithelial cells (BEAS-2B). DEP increased the expression of interleukin (IL)-6 and CXCL8. Silencing of the aryl hydrocarbon receptor (AhR) by siRNA or pretreatment with AhR-antagonists did not attenuate DEP-induced IL-6 and CXCL8 responses. However, the halogenated aromatic hydrocarbon (HAH)-selective AhR antagonist CH223191 caused a considerable reduction in DEP-induced CYP1A1 expression indicating that this response may be due to dioxin or dioxin-like constituents in DEP. Knock-down of protease activated receptor (PAR)-2 attenuated IL-6 responses without affecting CXCL8. Antioxidants did not affect IL-6 expression after 4h DEP-exposure and only partly reduced CXCL8 expression. However, after 24h exposure antioxidant treatment partly suppressed IL-6 protein release and completely blocked CXCL8 release. Furthermore, a heptane-soluble (non-polar) extract of DEP induced both IL-6 and CXCL8 release, whereas a PBS-soluble (highly polar) extract induced only IL-6. Thus, pro-inflammatory responses in DEP-exposed epithelial cells appear to be the result of both reactive oxygen species and receptor signaling, mediated through combinatorial effects between both non-polar and polar constituents adhered to the particle surface.

International audience; The mycotoxin alternariol (AOH), a frequent contaminant in fruit and cereal products, is known to induce DNA damage with subsequent cell cycle arrest. Here we elucidated the effects of AOH on stages of cell cycle progression using the RAW 264.7 macrophage model. AOH resulted in an accumulation of cells in the G2/M-phase (4N). Most cells exhibited a large G2 nucleus whereas numbers of true mitotic cells were reduced relative to control. Both cyclin B1 and p-cdc2 levels increased, while cyclin B1 remained in the cytoplasm; suggesting arrest in the G2/M transition point. Remarkably, after exposure to AOH for 24h, most of the cells exhibited abnormally shaped nuclei, as evidenced by partly divided nuclei, nuclear blebs, polyploidy and micronuclei (MN). AOH treatment also induced abnormal Aurora B bridges, suggesting that cytokinesis was interfered within cells undergoing karyokinesis. A minor part of the resultant G1 tetraploid (4N) cells re-entered the S-phase and progressed to 8N cells.

We have previously observed that 1-nitropyrene (1-NP) and its amine metabolite 1-aminopyrene (1-AP) induce differential chemokine responses in human bronchial epithelial cells (BEAS-2B) characterized by maximum responses for CXCL8 (IL-8) and CCL5 (RANTES), respectively. In the present study, we further explored the effects of 1-NP and 1-AP on chemokine responses. The results suggest that the differential effect of 1-NP and 1-AP on CXCL8 and CCL5 in BEAS-2B cells was mainly related to effects at higher concentrations, which in the case of 1-NP seemed to be linked to ROS-formation and/or metabolic activation by CYP-enzymes. However, at a low concentration (1 μM) where neither 1-NP, 1-AP nor unsubstituted pyrene had any effect on chemokine responses, we found that all three PAHs potentiated CXCL8 and CCL5 responses induced by the TLR3 ligand polyinosinic:polycytidylic acid (Poly I:C) in BEAS-2B cells. As neither benzo[a]pyrene nor β-naphthoflavone induced a similar effect in Poly I:C-primed cells, the response seemed independent of aryl hydrocarbon receptor-mediated mechanisms. The results show that priming cells with an inflammogenic stimuli like Poly I:C sensitizes the cells toward additional pro-inflammatory effects of certain PAHs. The study underscores that testing on healthy cells or animals may not be sufficient to fully evaluate chemokine responses and the pro-inflammatory potential of organic chemicals.

1-nitropyrene (1-NP), a common PAH in diesel exhaust, and its amine metabolite 1-aminopyrene (1-AP) induce distinctly different chemokine-responses in bronchial epithelial cells (BEAS-2B) characterized by increases in CXCL8 and CCL5, respectively. Tumor necrosis factor-α converting enzyme (TACE), which cleaves membrane-bound transforming growth factor (TGF)-α, activating the epidermal growth factor receptor (EGFR), may regulate pro-inflammatory responses induced by a variety of endogenous and exogenous agents. The present results suggest that CXCL8, but not CCL5 responses in 1-NP- or 1-AP-exposed cells required TACE/TGF-α/EGFR-signaling. The findings strengthen the notion that TACE/TGF-α/EGFR-signaling is central in epithelial CXCL8-regulation upon exposure to multiple airborne pollutants.

Diesel exhaust and its particles (DEP) have been under scrutiny for health effects in humans. In the development of these effects inflammation is regarded as a key process. Overall,in vitrostudies report similar DEP-induced changes in markers of inflammation, including cytokines and chemokines, as studiesin vivo.In vitrostudies suggest that soluble extracts of DEP have the greatest impact on the expression and release of proinflammatory markers. Main DEP mediators of effects have still not been identified and are difficult to find, as fuel and engine technology developments lead to continuously altered characteristics of emissions. Involved mechanisms remain somewhat unclear. DEP extracts appear to comprise components that are able to activate various membrane and cytosolic receptors. Through interactions with receptors, ion channels, and phosphorylation enzymes, molecules in the particle extract will trigger various cell signaling pathways that may lead to the release of inflammatory markers directly or indirectly by causing cell death.In vitrostudies represent a fast and convenient system which may have implications for technology development. Furthermore, knowledge regarding how particles elicit their effects may contribute to understanding of DEP-induced health effectsin vivo, with possible implications for identifying susceptible groups of people and effect biomarkers.

International audience; 1-Nitropyrene (1-NP) is a nitro-polycyclic aromatic hydrocarbon (nitro-PAH) present in diesel exhaust and bound to particular matter in urban air. We show that 1-NP and the referent PAH benzo(a)pyrene (BP) induce apoptosis and a lipid accumulation dependent on cytochrome P450 1A1-metabolites in mouse hepatoma cells, whereas 1-amino-pyrene had no effect. The caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-fmk), inhibits 1-NP-induced apoptosis, but failed to alter 1-NP-triggered lipid accumulation determined by Nile red staining. We further show that cholesterol and fatty acid contents are modified after nitro-PAH exposure and that 1-NP-induced cholesterol level is partially involved in related apoptosis. In parallel, the activity of the stearoyl-CoA desaturase 1 (SCD1), determined by fatty acid analysis, and its expression are reduced by 1-NP. The role of SCD1 in 1-NP-induced apoptosis is demonstrated in cells down-expressing SCD1, in which an increased apoptosis is observed, whereas the SCD1 overexpression elicits the opposite effects. In contrast, changes in SCD1 gene expression have no effect on the induced lipid accumulation. Moreover, 1-NP increases the activity of the AMP-dependent protein kinase (AMPK) leading to a caspase-independent apoptosis. Overall, our study demonstrates that the 1-NP-induced apoptosis is caspase- and AMPK-dependent, and is associated to a decrease of SCD1 expression which results in an alteration of lipid homeostasis.

Culturable mold in indoor air and its association with moisture-related problems and asthma and allergy among Swedish children

International audience; In this study, we show that the environmental pollutant, 3-nitrofluoranthene (3-NF) but not its amine form, 3-aminofluoranthene (3-AF), induces apoptosis as well as regulated necrosis with necroptotic features in Hepa1c1c7 cells. Upon exposure to 3-NF, both typical apoptotic and necrotic cells were observed. A large number of the cells exhibited a characteristic partial nuclear chromatin condensation. Cycloheximide completely attenuated 3-NF-induced cell death. Activation of caspase-8, -9, and -3 were observed. Moreover, Z-VAD-FMK decreased the apoptotic cells, whereas the number of propidium iodide (PI)-positive cells with partial chromatin condensation was reduced by Nec-1, an inhibitor of receptor interacting protein (RIP-1). Cyp1a1, but not nitric oxide synthase (NOS), appears to be involved in activation of 3-NF to reactive metabolites. Increase in the number as well as size of lysosomes, myelinosomes, and activation of autophagy were also observed. 3-NF induced phosphorylation of ERK1/2, JNK and p38 MAPKs. Interestingly, while inhibitors of ERK1/2 and JNK reduced apoptotic as well as necrotic cell death, the p38 inhibitor, SB202190 reduced only the necrotic cell death. Taken together, 3-NF elicits both apoptosis and a caspase-independent programmed cell death (PCD) with autophagic characteristics. Conversely, with 3-AF, no apparent cytotoxic effects besides a reduction in cell proliferation was observed.

Objectives Methacrylate monomers have been identified in aqueous extracts of freshly cured compomers. Both cells in the pulpal cavity and various cells of the oral mucosa can potentially be exposed to these leachables. Short-term exposure to dental monomers at relatively high concentrations induces adverse biological effects in vitro . The mechanisms involved have not been fully elucidated although involvement of various signaling pathways including ROS formation, activation of MAP-kinases and caspases has been suggested. The aim of this study was to investigate potential cellular responses following long-term exposure to relatively low and potentially more clinical relevant HEMA concentrations. Methods A submandibular gland cell line was exposed to HEMA (20–600 μM) for up to 72 h. The impact on cell proliferation, apoptosis, and possible underlying mechanisms was assessed by flow cytometry, microscopy and western blotting. Results Exposure to HEMA (600 μM) resulted in reduced cell proliferation after 24 h and increased apoptosis after 60 h. Further, we observed ATM dependent phosphorylation of p53, advocating an initial DNA damage in the HEMA exposed cells. Significance In conclusion, we show that exposure to relatively low concentration of HEMA for a prolonged time result in cell death, possibly as a consequence of DNA damage.

Nitrated-polycyclic aromatic hydrocarbons (nitro-PAHs) and diesel exhaust particle extracts (DEPE) induced apoptosis in Hepa1c1c7 cells with the following potency: 1,3-dinitropyrene (1,3-DNP)>1-nitropyrene (1-NP) >> DEPE >> 1,8-dinitropyrene (1,8-DNP). The compounds induced cyp1a1, and activated various intracellular signalling pathways related to apoptosis. The CYP inhibitor alpha-naphthoflavone strongly reduced 1,3-DNP-induced cell death, whereas cell death induced by 1-NP was rather increased. Toxic 1,3-DNP and 1-NP were found to induce a concentration-dependent lipid peroxidation. 1,3-DNP caused pro-apoptotic events, including increased phosphorylation and accumulation of p53 in the nucleus, cleavage of bid and of caspases 8 and 3, down-regulation of bcl-x(L) and phosphorylation of p38 and JNK MAPK. Furthermore, 1,3-DNP increased the activation of survival signals including phosphorylation of Akt and inactivation (phosphorylation) of pro-apoptotic bad. Although less potent, rather similar effects were observed following exposure to DEPE, compared to 1-NP. The most important finding was that the most mutagenic and carcinogenic compound tested, 1,8-DNP, induced little (if any) cell death, despite the fact that this compound seemed to give the most DNA damage as judged by DNA adduct formation, increased phosphorylation of p53 and accumulation of cells in S-phase. Immunocytochemical studies revealed that the p53 protein did not accumulate into the nucleus suggesting that 1,8-DNP inactivated the pro-apoptotic function of the p53 protein by a non-mutagenic event. These results suggest that after exposure to 1,8-DNP more cells may survive with DNA damage, thereby increasing its mutagenic and carcinogenic potential. The study has been supported in part by The Research Council of Norway, by Aurora grant (Egide) and Cancer Research UK; and Volker M. Arlt is a member of the European ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility) Network of Excellence. The technical assistance of Leni Johanne Ekeren and Trine Isaksen is very much appreciated. We also thank Dr. Trine Husøy for help with immunocytochemistry and Dr. Per E. Schwarze for support and critical reading of the manuscript.

Cadmium acetate (CdAc) induced apoptosis in primary alveolar type 2 cells and Clara cells from rat lung. Phosphorylation of the MAPKs ERK1/2, p38 and JNK was markedly increased in both cell types 15 min to 2 h after start of exposure to 10 microM CdAc. The phosphorylation of all the MAPKs remained elevated or was progressively increased up to 12 h. The p38 inhibitor SB202190 reduced the Cd-induced apoptosis, whereas the ERK and JNK inhibitors, PD98059 and JNKI1, respectively, did not have any significant effect. The activity of total PKC and the isoforms PKC(alpha) and PKC(delta) seemed initially to be high in type 2 cells and Clara cells. Exposure to 10 microM CdAc did not further increase the total PKC activity or phosphorylation levels of the specific isoforms. However, the PKC inhibitors, GF109203X and rottlerin partially reduced the Cd-induced apoptosis. Furthermore, exposure to GF109203X reduced the phosphorylation of p38 in Clara cells. In conclusion, the MAPK p38 seemed to be involved in the Cd-induced apoptosis in Clara cells and type 2 cells. The activity of PKC isoforms is suggested to have a permissive role in the apoptotic process, located upstream of p38 phosphorylation.

The reactive metabolites of benzo[a]pyrene (B[a]P) and cyclopenta[c,d]pyrene (CPP) induced an accumulation/phosphorylation of p53 in Hepa1c1c7 cells, whereas inhibition of p53 reduced the apoptosis. Judged by the inhibiting effect of wortmannin, phosphatidyl-inositol-3 (PI-3) kinases such as DNA-dependent protein kinase (DNA-PK), ATM (ataxia-telangiectasia mutated), and/or ATR (ATM related kinase), appeared to be involved in the DNA damage recognition and the B[a]P-/CPP-induced accumulation of p53. B[a]P and CPP also induced phosphorylation of jun-N-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). While inhibition of JNK had no effects on the B[a]P-/CPP-induced apoptosis, inhibition of p38 MAPK activity reduced this effect. Interestingly, survival signals such as phosphorylation of Akt and Bad seemed to be induced by the B[a]P-/CPP-compounds. Furthermore, also extracellular signal-regulated kinase (ERK)1/2 was activated and seemed to function as a survival signal in B[a]P-/CPP-induced apoptosis.

T-2 and HT-2 toxins belong to a group of mycotoxins that are widely encountered as natural contaminants known to elicit toxic responses in hematopoietic cells. In the present study, HL-60 cells were used to characterize the apoptotic effects of T-2 and a major metabolite, HT-2, and to examine the mechanisms involved. Apoptotic cells were identified microscopically by chromatin condensation and nuclear fragmentation, by flow cytometric analysis, and by DNA gel electrophoresis. T-2 and HT-2 induced concentration-dependent apoptosis after 24 h in HL-60 cells, starting at concentrations of 3.1 and 6.25 ng/ml respectively. An increased number of apoptotic cells could be observed 4–6 h after exposure to 12.5 ng/ml of toxin. Little cytotoxicity (plasma membrane damage) was observed even after exposure to concentrations of toxins (25–50 ng/ml) inducing apoptosis in 60–100% of the cells. The apoptotic process was almost completely blocked in the presence of the general caspase inhibitor zVAD.fmk. In contrast, no or only minor effects were observed with the more specific caspase inhibitors DEVD.CHO, IETD.fmk, and DEVD.fmk. As judged by Western blotting, the levels of several procaspases (-3, -7, -8, -9, but not -12) were reduced 3–6 h after exposure to toxin. Substantial increases in the presumed active form(s) of caspase-8 and -9 were observed. Furthermore, poly(ADP-ribose) polymerase (PARP) was already markedly cleaved 3 h after toxin treatment, indicative of active caspase-3 and -7. No or only minor changes in Bcl-2, Bcl-XL and Bax levels were observed. BAPTA-AM and ZnCl2 blocked the degradation of procaspases, the fragmentation of PARP, and the induction of apoptosis. In summary, both T-2 and HT-2 induced apoptosis, with T-2 being somewhat more potent than HT-2. The divalent calcium concentration, [Ca2+], appears to be involved in the activation of several caspases, resulting in DNA fragmentation, chromosomal condensation, and nuclear fragmentation.

A large number of halogenated phenols are detected in the blood of humans, fish and wild-animals. We have characterized the estrogen-like activity of phenol, 4-bromophenol (4-BP), 2,4-dibromophenol (2,4-DBP), 2,4,6-tribromophenol (2,4,6-TBP) and 4- tert -butylphenol ( tert -BP) using the estrogen-dependent human breast cancer cell line MCF-7. 4-BP, 2,4-DBP and 4- tert -BP all bind to the estrogen receptor (ER) with approximately 10 000-fold less affinity than 17β-estradiol (17β-E). 2,4,6-TBP was only able to displace 43% of radiolabelled estrogen when tested at concentrations up to 1 μM, whereas phenol had no affinity for the ER. 4- tert -BP stimulated cell growth and induced estrogen-regulated proteins such as the progesterone receptor (PgR) and pS2. The brominated phenols, however, although binding to the ER, did not stimulate cell growth or increase the levels of the PgR or pS2, or reduce the level of 17β-E induced pS2. On the contrary, 4-BP, 2,4-DBP and partly 4- tert -BP reduced 17β-E-stimulated cell growth apparently by an ER independent mechanism.

The usual starting points for hazard characterisation are No Observed Adverse Effect Levels (NOAELs)/benchmark doses for threshold effects and risk-specific doses/unit risks for non-threshold effects. In vitro studies are in general of no use in identifying these doses. However, based in part on in vitro investigations toxic equivalency factors have been developed for selected halogenated organic PCDD/PCDF/PCB congeners. Such factors can be used to determine the total toxic equivalent doses of mixtures of these contaminants. Studies with paracetamol illustrate that in vitro systems may help in the identification of the most sensitive species and strain. In vitro methods have been successfully used to studying qualitative and quantitative species differences in the toxicity of agents such as peroxisome proliferators and dichloromethane. Investigations with a number of chemicals show that in vitro systems are excellent models for characterisation of the mode of action of chemicals, but in vitro findings need to be validated in vivo. Experiments with bis(tri-n-butyltin)oxide illustrate that in vitro systems may aid in the extrapolation from high to low dose and from experimental animals to humans. In addition, in vitro approaches can be used to obtain useful information on the disposition of xenobiotics. It is concluded that if sufficient in vivo mechanistic information is available, in vitro studies using sub-cellular fractions/cells/tissue from animals and humans may significantly aid in the hazard characterisation of chemicals.

Tetrabromobisphenol A (TeBBPA) is a four-meta-brominated variant of bisphenol A (BPA) and is one of the most commonly used brominated flame retardants worldwide. We compared the estrogenic potency of TeBBPA, BPA and the brominated analogs mono- (MBBPA), di- (DBBPA), and tribromobisphenol A (TrBBPA) in the estrogen-dependent human breast cancer cell line MCF-7. All of the compounds competed with 17β-estradiol for binding to the estrogen receptor, although the affinity of the test chemicals to the estrogen receptor was much lower than that of 17β-estradiol. TrBBPA and TeBBPA showed a considerably lower access to the estrogen receptors within intact MCF-7 cells incubated in 100% serum compared to incubation in serum-free medium, indicating a strong binding to serum proteins. BPA, MBBPA, and DBBPA showed only a slightly reduced access to the receptors. All of the test compounds induced proliferation in MCF-7 cells, the potential decreasing with increasing number of bromo-substitutions. TeBBPA did not induce maximal cell growth, indicating cytotoxic effects at high concentrations. BPA and the brominated analogs, except TeBBPA, induced progesterone receptor and pS2 to the same extent as 17β-estradiol, although at much higher concentrations. Our studies demonstrate that compared to 17β-estradiol, BPA and the brominated analogs have much lower estrogenic potencies for all of the endpoints tested, TeBBPA being the least estrogenic compound.

The biotransformation of benz[j]aceanthrylene (B[j]A) was studied in suspensions of hepatocytes isolated from Aroclor 1254-treated or untreated rats. Using radiolabeled cofactors and metabolic inhibitors combined with UV, mass and 1H-NMR spectroscopy, we have detected five known metabolites and characterized nine new metabolites: metabolite 1 was tentatively assigned as B[j]A-1,2-dihydrodiol-8-sulfate; metabolite 2, B[j]A-1,2,9,10-tetrahydrotetrol; metabolite 3, B[j]A-1,2-dihydrodiol-10-O-glucuronide; metabolite 4, B[j]A-1-one-8-sulfate; metabolite 5, B[j]A-1,2-dihydrodiol-10-sulfate; metabolite 6, the sulfate conjugate of B[j]A-dihydrodiol-phenol; peak 7 in the chromatogram is a mixture of one glutathione conjugate and two sulfate conjugates of a B[j]A-metabolite; metabolite 8, B[j]A-10-O-glucuronide; metabolite 8', B[j]A-1,2-dihydrodiol; metabolite 9, B[j]A-10-sulfate; metabolite 9', B[j]A-9,10-dihydrodiol and metabolite 10, B[j]A-9,10-dihydro-9-hydroxy-10-sulfate. The metabolites identified support the notion that epoxidation at the cyclopenta region is an important activation step of B[j]A. Furthermore, sulfation appears to play a very important role in the conversion of hydroxylated B[j]A metabolites into more polar excretable products.

The genotoxicity of paracetamol, including covalent binding to DNA, induction of DNA single-strand breaks (SSBs), and inhibition of replicative and repair synthesis of DNA, has been investigated in rodents in vivo. In the covalent binding studies male ICR mice were fasted and pretreated with diethyl maleate to deplete hepatic glutathione (GSH) and 300 mg/kg of [G-3H]paracetamol was administered intraperitoneally (i.p.). Animals were killed at 2, 6, 24, 72 and 168 h after paracetamol and hepatic or renal DNA and protein were isolated and the extent of covalent binding determined. Maximal binding to liver DNA, 8.4 +/- 3.1 pmol/mg of DNA, was observed at 2 h and declined rapidly to 2.6 pmol/mg at 24 h. Measurable binding (1.4 pmol/mg of DNA) was detected at 7 days. Protein binding in the liver in these animals peaked between 2 and 6 h (887 pmol/mg of protein at 2 h) and declined monoexponentially to 52 pmol/mg at 7 days. Although based on a limited body of data, covalent binding was also detected in DNA isolated from the kidney. DNA damage measured as SSBs by alkaline elution was induced in nuclear DNA isolated from the liver but not from the kidney, 2 h after i.p. injection of paracetamol at 600 mg/kg in male B6 mice. Only marginal DNA damage was noted at 300 mg/kg. The alkaline elution profile from damaged liver nuclei was markedly biphasic, suggesting that breaks were induced in DNA from a subpopulation of liver cells. The non-hepatotoxic paracetamol regioisomer, acetyl-m-aminophenol (600 mg/kg), which binds covalently to proteins, did not cause DNA SSBs.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are mutagenic and carcinogenic environmental pollutants found in diesel exhaust and on urban air pollution particles. In the present study, human bronchial epithelial BEAS-2B cells were exposed to 2-nitrobenzanthrone (2-NBA) and 3-nitrobenzanthrone (3-NBA). DNA damage responses were compared to those observed after exposure to 1-nitropyrene (1-NP) and benzo[a]pyrene (B[a]P). Examination by microscopy revealed that 3-NBA was the most potent toxic compound while weaker responses were observed with 1-NP and B[a]P. Most interestingly, 2-NBA did not induce cell death or any other stress-related responses. 3-NBA induced a typical apoptotic cell death judged by nuclear condensation and little plasma membrane damage as well as cleavage of caspase 3 and poly-(ADP-ribose) polymerase (PARP). Exposure to 3-NBA resulted in an accumulation of cells in S-phase, and further analysis by Western blotting, immunocytochemistry and flow cytometry revealed that 3-NBA induced a DNA damage response characterized by phosphorylation of ATM (ataxia-telangiectasia mutated), checkpoint kinase (Chk) 2/Chk1, H2AX and p53. The p53 inhibitor pifithrin-α inhibited 3-NBA-induced apoptosis while small effects were seen using pifithrin-μ, suggesting that 3-NBA-induced cell death is a result of transcriptional activation of p53. In conclusion, 3-NBA is a potent inducer of apoptosis, which seemed to be triggered by the DNA damage response. Furthermore, a change of the nitro-group to the second position (i.e. 2-NBA) dramatically changed the cellular reactivity of the compound.

Benz[j]aceanthrylene (B[j]A) and benz[l]aceanthrylene (B[l]A), two isomeric cyclopenta polycyclic aromatic hydrocarbons (CP-PAH) structurally related to 3-methylcholanthrene, were studied with respect to their genotoxic effects in isolated liver and lung cells. Both compounds were found to cause DNA adducts measured by the 32P-postlabelling technique. The level of DNA-adducts in rat hepatocytes exposed to 30 micrograms/ml B[l]A and B[j]A for 4 h were 46.5 +/- 22.0 and 8.3 +/- 5.1 fmol/micrograms DNA respectively. Using butanol extractions, the major and one of the minor B[j]A adducts co-chromatographed with B[j]A-1,2-oxide adducts of 2'-deoxyadenosine and 2'-deoxyguanosine. Thus, oxidation at the cyclopenta-ring of B[j]A appears to be an important activation pathway. In hepatocytes, 3-30 micrograms/ml of B[j]A and B[l]A induced DNA damage and repair measured both as increased alkaline elution of DNA and as increased incorporation of [3H]TdR in the DNA. B[l]A was somewhat more potent than B[j]A in inducing DNA repair. Reactive CP-PAH intermediates formed in the hepatocytes caused mutations in Salmonella typhimurium TA98 upon co-incubation. DNA adducts were also observed in isolated rabbit lung cells exposed to 30 micrograms/ml B[l]A or B[j]A for 2 h. A total of 14.5 +/- 6.9, 2.9 +/- 2.1 and 0.2 +/- 0.6 fmol B[l]A adducts/micrograms DNA were observed in Clara cells, type II pneumocytes and alveolar macrophages respectively. The main B[l]A adduct observed in the liver cells was not found in the lung cells. On the other hand, the levels of B[j]A adducts in the lung cells were in the range 4-14% of that found in liver cells, and no major differences between the various lung cells were observed. Neither B[l]A nor B[j]A induced DNA damage measured by alkaline elution in the lung cells, indicating that these adducts are not alkali labile.

International audience; Polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs are environmental pollutants formed during incomplete combustion of organic material. Many possess mutagenic and carcinogenic properties, but their effects on cell death are less known. We have found that rather similar PAHs cause death by quite different mechanisms including apoptosis, necrosis and various mixtures of the two. In this addendum to our recent publication, Toxicology 2009; 255:140-50, we report that 3-nitrofluoranthene (3-NF) induces apoptosis as well as regulated necrosis with necroptotic features. The typical necroptotic cell exhibited partial nuclear chromatin condensation combined with damaged plasma membrane. The cells were characterized by increased size as well as number of lysosomes and myelinosomes/autophagic vesicles, and also in expression of the autophagic marker, LC3B. However, the induced autophagy appears to be a parallel event rather than the cause of cell death.

1,2-Dibromo-3-chloropropane (DBCP) induced DNA damage, measured by alkaline elution, at low concentrations (5-10 microM) in suspensions of hepatocytes and testicular cells isolated from rats. At higher concentrations (greater than or equal to 100 microM) DBCP caused DNA damage and increased the frequency of sister chromatid exchanges in Chinese hamster V79 cells. When DBCP (2.5-10 microM) was tested for its ability to cause unscheduled DNA synthesis (UDS) in monolayers of liver cells isolated from untreated rats, a clear positive response was obtained. No increase in UDS was detectable when liver cells isolated from PCB-pretreated rats were used. In contrast, DBCP (greater than or equal to 50 microM) was metabolized in hepatocytes from PCB-pretreated rats to products mutagenic to Salmonella typhimurium TA100 in co-culture with the hepatocytes, whereas control hepatocytes were substantially (40-fold) less active. No bacterial mutagenicity could be detected when the TA100 strain was co-incubated with isolated rat testicular cells and DBCP. In co-cultures of hepatocytes and V79 cells DBCP-induced DNA damage in V79 cells occurred at low concentrations (10 microM DBCP) compared to the concentration (100 microM) needed to induce DNA damage in the V79 cells incubated without hepatocytes. Testicular cells were not able to enhance DBCP-induced DNA damage in the V79 cells. The data indicate that the putative reactive DBCP episulphonium ion metabolite formed in the testicular cells presumably responsible for testicular cell DNA damage, is not capable of escaping the cell where it is formed. Other reactive DBCP metabolites generated in the liver cells seem to be able to interact with the DNA of neighbouring cells.

Effects of paracetamol have been studied in a hydroxyurea (HU)-resistant mouse mammary tumour cell line TA3H2, shown to overproduce the small subunit of ribonucleotide reductase. These TA3H2 cells were much more resistant than the TA3H (wild-type) cells towards the inhibitory effect of paracetamol on cell growth, IC50 0.55 mM paracetamol for the wild-type compared to 2.7 mM for the HU-resistant cells. The reduced cell growth was due to an inhibition of replicative DNA synthesis, judged from an increased percentage of cells in S-phase measured by flow cytometry. Furthermore, in the wild-type cells, the increase in the number of cells in S phase was already observed at 0.1 mM while in the HU-resistant cell line this effect was first seen at 3.0 mM paracetamol. HU inhibits ribonucleotide reductase by destroying a tyrosyl free radical located on the small subunit of the enzyme. By electron paramagnetic resonance we demonstrate that paracetamol added to crude cell extracts of HU-resistant cells also immediately destroys this radical. These results show that paracetamol reduces DNA synthesis by a specific inhibition of ribonucleotide reductase. A concentration-dependent induction of sister chromatid exchanges was found both with paracetamol (1.0-10 mM) and HU (0.3-3 mM) in wild-type cells whereas no such increase was observed in HU-resistant cells. Paracetamol (1 mM for 2 h) also increased the number of chromosomal aberrations CAs in wild-type cells (i.e. chromatid breaks and chromatid exchanges). The frequency of CAs was not increased in HU-resistant cells at paracetamol concentrations up to 10 mM.(ABSTRACT TRUNCATED AT 250 WORDS).

In the present study, possible mechanisms involved in fluoride-induced apoptosis in a human epithelial lung cell line (A549) were examined. Sodium fluoride (NaF) induced apoptosis in the A549 cells, with a maximum at 5-7.5 mM after 20 hours of exposure. The number of cells with plasma membrane damage (PI-positive cells) increased moderately up to 5 mM, but markedly at 7.5 mM. Deferoxamine (an Al3chelator) almost completely prevented these NaF-induced responses, which may suggest a role for G protein activation. The apoptotic effect was partially reduced by the PKA inhibitor H89. NaF induced a weak but sustained increase in PKC activity, whereas the PKC activator TPA induced a transient effect. TPA, which enhanced the NaF-induced PKC activity, was not apoptotic when added alone, but facilitated the NaFinduced apoptosis and the increase in PI-positive cells. PKC downregulation induced by TPA pretreatment almost completely prevented the NaF-induced apoptosis and the increase in PI-positive cells. Pretreatment with the PKC inhibitor GF109203X, which abolished the PKC activity after 3 hours, enhanced the NaF-induced apoptosis. KN93 (a CaM kinase II inhibitor) and W7 (a calmodulin inhibitor) seem to reduce the apoptotic effect of NaF, whereas BAPTA-AM (a Ca2chelator) was without effect. The tyrosine kinase inhibitor genistein also markedly reduced the NaF-induced apoptosis, whereas the PI-3 kinase inhibitor wortmannin augmented the response. In conclusion, the present results suggest that NaF induces an apoptotic effect and an increase in PI-positive A549 cells via similar mechanisms, involving PKC, PKA, tyrosine kinase and Ca2linked enzymes, whereas PI-3 kinase seems to exert a counteracting effect.

The effects of paracetamol on the repair of DNA damage in resting human peripheral mononuclear blood cells (MNC) in vitro were investigated by means of the alkaline elution technique. Low doses of UV light (254 nm, 3 J/m2) caused a transient increase in the amount of DNA single-strand breaks and alkali-labile sites (SSBs). Paracetamol (0.1-1.0 mM) present during post-irradiation incubation approximately doubled the maximum level of UV-induced (1-3 J/m2) SSBs and delayed the completion of repair. Although there were considerable variations between cells prepared from different donors, the level of UV-induced DNA SSBs was always higher with paracetamol. Hydroxyurea (0.3 mM), an inhibitor of ribonucleotide reductase, caused a similar increased accumulation and slow removal of SSBs, whereas cytosine-1-beta-D-arabinofuranoside (Ara C) (10 microM), an inhibitor of DNA polymerases, led to a steady accumulation of DNA SSBs. The increased levels of SSBs caused by paracetamol or hydroxyurea were both completely suppressed by concomitant addition of deoxyribonucleosides; this supports the notion that paracetamol as well as hydroxyurea inhibits ribonucleotide reductase. About the same rates of formation and removal of UV-induced SSBs were observed in T lymphocytes, B lymphocytes and monocytes. In both isolated T lymphocytes and B lymphocytes, paracetamol (0.3 mM) markedly increased the level of DNA SSBs induced by UV, whereas monocytes seemed to be less sensitive to the effect of paracetamol. It is concluded that the inhibition of DNA repair may contribute to the clastogenic effects of paracetamol.

1,2-Dibromo-3-chloropropane (DBCP) induced DNA damage, measured by an automated alkaline elution method, in suspensions of rat liver parenchymal cells at low concentrations (1-10 microM). At much higher concentrations (0.5-2.5 mM), DBCP was metabolized to products that were mutagenic to Salmonella typhimurium TA100 co-incubated with the liver cells. At these higher concentrations a marked depletion of cellular glutathione was seen and at 2.5 mM DBCP was cytotoxic. Perdeuterated DBCP (D5-DBCP) caused less DNA damage in the liver cells than DBCP, most likely because of decrease in cytochrome P-450 dependent metabolism. A more pronounced decrease in mutagenicity occurred with D5-DBCP compared to DBCP, whereas the two compounds were equally cytotoxic. Preincubation of the liver cells with diethylmaleate or buthionine sulfoximine, to lower cellular levels of glutathione, decreased DBCP induced DNA damage. The decrease in DNA damage was proportional to the decrease in cellular glutathione levels. In contrast, diethylmaleate enhanced DBCP-induced bacterial mutagenicity and cellular cytotoxicity. The cytotoxic effect could be partly blocked by addition of ascorbate. From the data presented we suggest that: (i) cytochrome P-450 dependent oxidation as well as glutathione conjugation are involved in DBCP induced DNA damage, (ii) cytochrome P-450 dependent oxidation leads to formation of products mutagenic to bacteria and (iii) the cytotoxicity induced by DBCP in the liver cells in vitro is caused by oxidative damage following glutathione depletion and/or direct membrane damage.

Isolated rat hepatocytes (4 X 10(6) cells/ml metabolized the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP) (100 microM) to at least eight different metabolites in a 4 h duration. The major metabolite formed was 4-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate (4'-PhIP sulfate). Its rate of formation was increased in hepatocytes from PCB pretreated animals in comparison to hepatocytes from untreated animals. One of the other metabolites was the unconjugated derivative of the sulfate (4'-OH-PhIP). This metabolite was also found after in-vitro incubations of rat liver microsomes from PCB or beta-naphthoflavone pretreated animals. The evidence for the proposed structure of the major metabolite is based on [1H]NMR and UV spectroscopy, incorporation of radiolabelled sulfate and arylsulfatase-lability. The formation of 4'-PhIP sulfate was inhibited by the P-450 inhibitors alpha-naphthoflavone and metyrapone and when incubated in sulfate-free medium added the sulfotransferase inhibitor pentachlorophenol. 4'-PhIP sulfate was also the major metabolite of PhIP in the urine of exposed rat.

N-isopropyl-alpha-(2-methylhydrazino)-p-toluamide hydrochloride (procarbazine; 50-1000 micrograms/ml) induced DNA damage in hepatocytes measured by an automated alkaline elution method, whereas no significant increase in unscheduled DNA synthesis was seen. In hepatocytes isolated from PCB-treated rats, DNA damage was detected in both test systems at concentrations as low as 1-10 micrograms/ml. DNA damage, as measured by alkaline elution and sister-chromatid exchange(s), was observed also in V79 cells incubated with PCB-hepatocytes. In contrast, no mutagenic activity was observed in the Salmonella typhimurium strain TA1530 co-incubated with the hepatocytes. Exposure of rats to low doses of procarbazine (25-50 mg/kg) caused DNA damage measured by alkaline elution in liver and testis, with the liver being somewhat more sensitive. The genotoxicity caused by procarbazine was increased by a factor of 2-3 in both organs by PCB-treatment of the rats. N-isopropyl-alpha-(2-methyl-hydrazino)-p-[alpha,alpha-2H2]toluamide (d2-procarbazine), was found to cause significantly less genotoxicity in control rats than either procarbazine itself, or N-isopropyl-alpha-(2-[alpha,alpha,alpha-2H3]methylhydrazino)-p-tol uamide (d3-procarbazine). This indicates that benzylic C-H oxidation of procarbazine is an important step in the activation of procarbazine to genotoxic metabolites in uninduced rats.