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Inhalation of ambient particulate matter (PM) and ozone (O 3 ) has been associated with increased cardiovascular morbidity and mortality. However, the interactive effects of PM and O 3 on cardiac dysfunction and disease have not been thoroughly examined, especially at a proteomic level. The purpose of this study was to identify and compare proteome changes in spontaneously hypertensive (SH) rats co-exposed to concentrated ambient particulates (CAPs) and O 3 , with a focus on investigating inflammatory and metabolic pathways, which are the two major ones implicated in the pathophysiology of cardiac dysfunction. For this, we measured and compared changes in expression status of 9 critical pro- and anti-inflammatory cytokines using multiplexed ELISA and 450 metabolic proteins involved in ATP production, oxidative phosphorylation, cytoskeletal organization, and stress response using two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) in cardiac tissue of SH rats exposed to CAPs alone, O 3 alone, and CAPs + O 3 . Proteomic expression profiling revealed that CAPs alone, O 3 alone, and CAPs + O 3 differentially altered protein expression patterns, and utilized divergent mechanisms to affect inflammatory and metabolic pathways and responses. Ingenuity Pathway Analysis (IPA) of the proteomic data demonstrated that the metabolic protein network centered by gap junction alpha-1 protein (GJA 1) was interconnected with the inflammatory cytokine network centered by nuclear factor kappa beta (NF-kB) potentially suggesting inflammation-induced alterations in metabolic pathways, or vice versa, collectively contributing to the development of cardiac dysfunction in response to CAPs and O 3 exposure. These findings may enhance understanding of the pathophysiology of cardiac dysfunction induced by air pollution and provide testable hypotheses regarding mechanisms of action., Competing Interests: Declarations Conflict of interest The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Ethical Approval This manuscript has been reviewed by The Center for Computational Toxicology and Exposure, United States Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Epidemiological evidence suggests the potential for air pollutants to induce male reproductive toxicity. In experimental studies, exposure to ozone during sensitive windows in the sperm lifecycle has been associated with impaired sperm motility. Subsequently, we sought to investigate the effects of episodic exposure to ozone during sperm maturation in the rat. Long-Evans rats were exposed to either filtered air or ozone (0.4 or 0.8 ppm) for five non-consecutive days over two weeks. Ozone exposure did not impact male reproductive organ weights or sperm motility ∼24 hours following the final exposure. Furthermore, circulating sex hormones remained unchanged despite increased T 3 and T 4 in the 0.8 ppm group. While there was indication of altered adrenergic signaling attributable to ozone exposure in the testis, there were minimal impacts on small non-coding RNAs detected in cauda sperm. Only two piwi-interacting RNAs (piRNAs) were altered in the mature sperm of ozone-exposed rats (piR-rno-346434 and piR-rno-227431). Data across all rats were next analyzed to identify any non-coding RNAs that may be correlated with reduced sperm motility. A total of 7 microRNAs (miRNAs), 8 RNA fragments, and 1682 piRNAs correlated well with sperm motility. Utilizing our exposure paradigm herein, we were unable to substantiate the relationship between ozone exposure during maturation with sperm motility. However, these approaches served to identify a suite of non-coding RNAs that were associated with sperm motility in rats. With additional investigation, these RNAs may prove to have functional roles in the acquisition of motility or be unique biomarkers for male reproductive toxicity., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Corresponding author is co-editor for special issue on reproductive toxicology and air pollution and current sits on the editorial board - CM If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

Altered fetal growth, which can occur due to environmental stressors during pregnancy, may program a susceptibility to metabolic disease. Gestational exposure to the air pollutant ozone is associated with fetal growth restriction in humans and rodents. However, the impact of this early life ozone exposure on offspring metabolic risk has not yet been investigated. In this study, fetal growth restriction was induced by maternal inhalation of 0.8 ppm ozone on gestation days 5 and 6 (4 hr/day) in Long Evans rats. To uncover any metabolic inflexibility, or an impaired ability to respond to a high-fat diet (HFD), a subset of peri-adolescent male and female offspring from filtered air or ozone exposed dams were fed HFD (45% kcal from fat) for 3 days. By 6 weeks of age, male and female offspring from ozone-exposed dams were heavier than offspring from air controls. Furthermore, offspring from ozone-exposed dams had greater daily caloric consumption and reduced metabolic rate when fed HFD. In addition to energy imbalance, HFD-fed male offspring from ozone-exposed dams had dyslipidemia and increased adiposity, which was not evident in females. HFD consumption in males resulted in the activation of the protective 5'AMP-activated protein kinase (AMPKα) and sirtuin 1 (SIRT1) pathways in the liver, regardless of maternal exposure. Unlike males, ozone-exposed female offspring failed to activate these pathways, retaining hepatic triglycerides following HFD consumption that resulted in increased inflammatory gene expression and reduced insulin signaling genes. Taken together, maternal ozone exposure in early pregnancy programs impaired metabolic flexibility in offspring, which may increase susceptibility to obesity in males and hepatic dysfunction in females., (Published 2022. This article is a U.S. Government work and is in the public domain in the USA.)

Conazoles are fungicides used as agricultural pesticides and pharmaceutical products. We investigated whether a common core of toxicological and transcriptional responses underlies the observed carcinogenic effects of three conazoles: cyproconazole, epoxiconazole, and propiconazole. In studies where mice were fed diets of these conazoles for 30 days, we found a common set of toxicological effects altered by these conazoles: hepatomegaly, hepatocellular hypertrophy, decreased serum cholesterol, decreased hepatic levels of all-trans-retinoic acid, and increased hepatic cell proliferation. Microarray-based transcriptional analysis revealed 330 significantly altered probe sets common to these conazoles, many of which showed strong dose responses for cytochrome P450, glutathione S-transferase, and oxidative stress genes. More detailed analyses identified a subset of 80 altered genes common to the three conazoles that were associated with cancer. Pathways associated with these genes included xenobiotic metabolism, oxidative stress, cell signaling, and cell proliferation. A common TGFα-centric pathway was identified within the 80-gene set, which, in combination with the toxicological and other transcriptomic findings, provides a more refined toxicity profile for these carcinogenic conazoles.

Propiconazole induces hepatocellular carcinomas and hepatocellular adenomas in mice and promotes liver tumors in rats. Transcriptional, proteomic, metabolomic and biochemical studies of hepatic tissues from mice treated with propiconazole under the conditions of the chronic bioassay indicated that propiconazole induced oxidative stress. Here we sought to identify the source of the reactive oxygen species (ROS) induced by propiconazole using both AML12 immortalized mouse hepatocytes in culture and liver tissues from mice. We also sought to further characterize the nature and effects of ROS formation induced by propiconazole treatment in mouse liver. ROS was induced in AML12 cells by propiconazole as measured by fluorescence detection and its formation was ameliorated by N-acetylcysteine. Propiconazole induced glutathione-S-transferase (GSTα) protein levels and increased the levels of thiobarbituric acid reactive substances (TBARS) in AML12 cells. The TBARS levels were decreased by diphenylene iodonium chloride (DPIC), a cytochrome P450 (CYP) reductase inhibitor revealing the role of CYPs in ROS generation. It has been previously reported that Cyp2b and Cyp3a proteins were induced in mouse liver by propiconazole and that Cyp2b and Cyp3a proteins undergo uncoupling of their CYP catalytic cycle releasing ROS. Therefore, salicylic acid hydroxylation was used as probe for ROS formation using microsomes from mice treated with propiconazole. These studies showed that levels of 2,3-dihydroxybenzoic acid (an ROS derived metabolite) were decreased by ketoconazole, melatonin and DPIC. In vivo, propiconazole increased hepatic malondialdehyde levels and GSTα protein levels and had no effect on hepatic catalase or superoxide dismutase activities. Based on these observations we conclude that propiconazole induces ROS in mouse liver by increasing CYP protein levels leading to increased ROS levels. Our data also suggest that propiconazole induces the hydroxyl radical as a major ROS form., (Published by Elsevier Ireland Ltd.)

Propiconazole is a mouse hepatotumorigenic fungicide and has been the subject of recent investigations into its carcinogenic mechanism of action. The goals of this study were (1) to identify metabolomic changes induced in the liver by increasing doses of propiconazole in mice, (2) to interpret these results with key previously reported biochemical, transcriptomic, and proteomic findings obtained from mouse liver under the same treatment conditions, and (3) to relate these alterations to those associated with the carcinogenesis process. Propiconazole was administered to male CD-1 mice in the feed for 4 days with six mice per feed level (500, 1250, and 2500 ppm). The 2500 ppm dose level had previously been shown to induce both adenocarcinomas and adenomas in mouse liver after a 2-year continuous feed regimen. Endogenous biochemicals were profiled using liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry methods and 261 were detected. The most populous biochemical class detected was lipids, followed by amino acids and then carbohydrates. Nucleotides, cofactors and vitamins, energy, peptides, and xenobiotics were also represented. Of the biochemicals detected, 159 were significantly altered by at least one dose of propiconazole and many showed strong dose responses. Many alterations in the levels of biochemicals were found in the glycogen metabolism, glycolysis, lipolysis, carnitine, and the tricarboxylic acid cycle pathways Several groups of metabolomic responses were ascribed to the metabolism and clearance of propiconazole: glucuronate, glutathione, and cysteine pathways. Groups of metabolic responses supported previous hypotheses on key events that can lead to propiconazole-induced tumorigenesis: oxidative stress and increases in the cholesterol biosynthesis pathway. Groups of metabolomic responses identified biomarkers associated with neoplasia: increases in glycolysis and increases in the levels of spermidine, sarcosine, and pseudouridine. These results extended the companion transcriptomic and proteomic studies and provided a more complete understanding of propiconazole's effects in mouse liver.

Benzo[a]pyrene (B[a]P) is a potent human and rodent lung carcinogen. This activity has been ascribed in part to the formation of anti-trans-7,8-dihydroxy-7,8-dihydroB[a]P-9,10-epoxide (BPDE)-DNA adducts. Other carcinogenic mechanisms have been proposed: (1) the induction of apurinic sites from radical cation processes, and (2) the metabolic formation of B[a]P-7,8-quinone (BPQ) that can form covalent DNA adducts or reactive oxygen species which can damage DNA. The studies presented here sought to examine the role of stable BPQ-DNA adducts in B[a]P-induced mouse lung tumorigenesis. Male strain A/J mice were injected intraperitoneally once with BPQ or trans-7,8-dihydroxy-7,8-dihydroB[a]P (BP-7,8-diol) at 30, 10, 3, or 0mg/kg. Lungs and livers were harvested after 24h, the DNA extracted and subjected to (32)P-postlabeling analysis. Additional groups of mice were dosed once with BPQ or BP-7,8-diol each at 30 mg/kg and tissues harvested 48 and 72 h later, or with B[a]P (50mg/kg, a tumorigenic dose) and tissues harvested 72 h later. No BPQ or any other DNA adducts were observed in lung or liver tissues 24, 48, or 72 h after the treatment with 30 mg/kg BPQ. BP-7,8-diol gave BPDE-DNA adducts at all time points in both tissues and B[a]P treatment gave BPDE-DNA adducts in the lung. In each case, no BPQ-DNA adducts were detected. Mouse body weights significantly decreased over time after BPQ or BP-7,8-diol treatments suggesting that systemic toxicity was induced by both agents. Model studies with BPQ and N-acetylcysteine suggested that BPQ is rapidly inactivated by sulfhydryl-containing compounds and not available for DNA adduction. We conclude that under these treatment conditions BPQ does not form stable covalent DNA adducts in the lungs or livers of strain A/J mice, suggesting that stable BPQ-covalent adducts are not a part of the complex of mechanisms involved in B[a]P-induced mouse lung tumorigenesis., ((c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Conazoles are fungicides used in agriculture and as pharmaceuticals. In a previous toxicogenomic study of triazole-containing conazoles we found gene expression changes consistent with the alteration of the metabolism of all trans-retinoic acid (atRA), a vitamin A metabolite with cancer-preventative properties (Ward et al., Toxicol. Pathol. 2006; 34:863-78). The goals of this study were to examine effects of propiconazole, triadimefon, and myclobutanil, three triazole-containing conazoles, on the microsomal metabolism of atRA, the associated hepatic cytochrome P450 (P450) enzyme(s) involved in atRA metabolism, and their effects on hepatic atRA levels in vivo. The in vitro metabolism of atRA was quantitatively measured in liver microsomes from male CD-1 mice following four daily intraperitoneal injections of propiconazole (210 mg/kg/d), triadimefon (257 mg/kg/d) or myclobutanil (270 mg/kg/d). The formation of both 4-hydroxy-atRA and 4-oxo-atRA were significantly increased by all three conazoles. Propiconazole-induced microsomes possessed slightly greater metabolizing activities compared to myclobutanil-induced microsomes. Both propiconazole and triadimefon treatment induced greater formation of 4-hydroxy-atRA compared to myclobutanil treatment. Chemical and immuno-inhibition metabolism studies suggested that Cyp26a1, Cyp2b, and Cyp3a, but not Cyp1a1 proteins were involved in atRA metabolism. Cyp2b10/20 and Cyp3a11 genes were significantly over-expressed in the livers of both triadimefon- and propiconazole-treated mice while Cyp26a1, Cyp2c65 and Cyp1a2 genes were over-expressed in the livers of either triadimefon- or propiconazole-treated mice, and Cyp2b10/20 and Cyp3a13 genes were over-expressed in the livers of myclobutanil-treated mice. Western blot analyses indicated conazole induced-increases in Cyp2b and Cyp3a proteins. All three conazoles decreased hepatic atRA tissue levels ranging from 45-67%. The possible implications of these changes in hepatic atRA levels on cell proliferation in the mouse tumorigenesis process are discussed.

Benzo[a]pyrene-7,8-quinone (BPQ) is one of the reactive metabolites of the widely distributed archetypal polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P). The formation of BPQ from B[a]P through trans-7,8-dihydroxy-7,8-dihydroB[a]P by the mediation of aldo-keto reductases and its role in the genotoxicity and carcinogenesis of B[a]P currently are under extensive investigation. Toxicity pathways related to BPQ are believed to include both stable and unstable (depurinating) DNA adduct formation as well as reactive oxygen species. We previously reported the complete characterization of four novel stable BPQ-deoxyguanosine (dG) and two BPQ-deoxyadenosine (dA) adducts (Balu et al., Chem. Res. Toxicol. 17 (2004) 827-838). However, the identification of BPQ-DNA adducts by 32P postlabeling methods from in vitro and in vivo exposures required 3'-monophosphate derivatives of BPQ-dG, BPQ-dA, and BPQ-deoxycytidine (dC) as standards. Therefore, in the current study, BPQ adducts of dGMP(3'), dAMP(3'), and dCMP(3') were prepared. The syntheses of the BPQ-3'-mononucleotide standards were carried out in a manner similar to that reported previously for the nucleoside analogs. Reaction products were characterized by UV, LC/MS analyses, and one- and two-dimensional NMR techniques. The spectral studies indicated that all adducts existed as diastereomeric mixtures. Furthermore, the structural identities of the novel BPQ-dGMP, BPQ-dAMP, and BPQ-dCMP adducts were confirmed by acid phosphatase dephosphorylation of the BPQ-nucleotide adducts to the corresponding known BPQ-nucleoside adduct standards. The BPQ-dGMP, BPQ-dAMP, and BPQ-dCMP adduct standards were used in 32P postlabeling studies to identify BPQ adducts formed in vitro with calf thymus DNA and DNA homopolymers. 32P postlabeling analysis revealed the formation of 8 major and at least 10 minor calf thymus DNA adducts. Of these BPQ-DNA adducts, the following were identified: 1 BPQ-dGMP adduct, 2 BPQ-dAMP adducts, and 3 BPQ-dCMP adducts. This study represents the first reported example of the characterization of stable BPQ-DNA adducts in isolated mammalian DNA and is expected to contribute significantly to the future BPQ-DNA adduct studies in vivo and thereby to the contribution of BPQ in B[a]P carcinogenesis.

Benzo[a]pyrene (B[a]P) is an archetypal member of the family of polycyclic aromatic hydrocarbons (PAHs) and is a widely distributed environmental pollutant. B[a]P is known to induce cancer in animals, and B[a]P-containing complex mixtures are human carcinogens. B[a]P exerts its genotoxic and carcinogenic effects through metabolic activation forming reactive intermediates that damage DNA. DNA adduction by B[a]P is a complex phenomenon that involves the formation of both stable and unstable (depurinating) adducts. One pathway by which B[a]P can mediate genotoxicity is through the enzymatic formation of B[a]P-7,8-quinone (BPQ) from B[a]P-7,8-diol by members of the aldo-keto-reductase (AKR) family. Once formed, BPQ can act as a reactive Michael acceptor that can alkylate cellular nucleophiles including DNA and peptides. Earlier studies have reported on the formation of stable and depurinating adducts from the reaction of BPQ with DNA and nucleosides, respectively. However, the syntheses and characterization of the stable adducts from these interactions have not been addressed. In this study, the reactivity of BPQ toward 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) nucleosides under physiological pH conditions is examined. The identification and characterization of six novel BPQ-nucleoside adducts obtained from the reaction of BPQ and dG or dA in a mixture of phosphate buffer and dimethylformamide are reported. The structures of these adducts were determined by ultraviolet spectroscopy, electrospray mass spectrometry, and NMR experiments including (1)H, (13)C, two-dimensional COSY, one-dimensional NOE, ROESY, HMQC, HSQC, and HMBC. The reaction of BPQ with dG afforded four unique Michael addition products: two diastereomers of 8-N(1),9-N(2)-deoxyguanosyl-8,10-dihydroxy-9,10-dihydrobenzo[a]pyren-7(8H)-one (BPQ-dG(1,2)) and two diastereomers of 10-(N(2)-deoxyguanosyl)-9,10-dihydro-9-hydroxybenzo[a]pyrene-7,8-dione (BPQ-dG(3,4)). The BPQ-dG(1,2)( )()adducts suggest a 1,6-Michael addition reaction of dG, an oxidation of the hydroquinone to the quinone, a 1,4-Michael addition of water, and an internal cyclization. The BPQ-dG(3,4)( )()adducts suggest a 1,4-Michael addition reaction of dG, an oxidation of the hydroquinone to the quinone, and a 1,6-Michael addition of water. Under similar but extended reaction conditions, the reaction of BPQ with dA produced only one diastereomeric pair of adducts identified as 8-N(6),10-N(1)-deoxyadenosyl-8,9-dihydroxy-9,10-dihydrobenzo[a]pyren-7(8H)-one (BPQ-dA(1,2)). The BPQ-dA(1,2)( )()adducts suggest a 1,4-Michael addition reaction of dA, an oxidation of the hydroquinone to the quinone, a 1,6-Michael addition of water, and an internal cyclization. As considerable efforts have been placed in documenting the genotoxic effects of BPQ, this first report of the identification and characterization of these stable adducts of BPQ formed under physiological pH conditions is expected to contribute significantly to the area of BPQ-mediated genotoxicity and carcinogenesis.

The biotransformation of (+/-)-trans-4,5-dihydroxy-4, 5-dihydrobenzo[a]pyrene (trans-B[a]P-4,5-diol), the K-region dihydrodiol of B[a]P, by beta-naphthoflavone (BNF)-induced rat liver microsomes was studied. trans-B[a]P-4,5-diol was metabolized to six major products as characterized by NMR, MS, and UV spectroscopy, and all were identified as bis-diols: two diastereomers of trans,trans-4, 5:7,8-tetrahydroxy-4,5:7,8-tetrahydrobenzo[a]pyrene (trans, trans-B[a]P-4,5:7,8-bis-diol), two diastereomers of trans,trans-4, 5:9,10-tetrahydroxy-4,5:9,10-tetrahydrobenzo[a]pyrene (trans, trans-B[a]P-4,5:9,10-bis-diol), and two diastereomers of the somewhat unusual trans,trans-1,2:4,5-tetrahydroxy-1,2:4, 5-tetrahydrobenzo[a]pyrene (trans,trans-B[a]P-1,2:4,5-bis-diol). BNF-induced rat liver microsomes also metabolized B[a]P to the same trans-B[a]P-4,5-diol-derived bis-diols. The ability of trans-B[a]P-4, 5-diol to form DNA adducts was investigated using (32)P-postlabeling techniques specifically designed to detect stable polar DNA adducts. Four DNA adducts were detected after microsomal activation of trans-B[a]P-4,5-diol with calf thymus DNA. Further analyses indicated that each of these stable polar DNA adducts was derived from the further metabolic activation of the trans,trans-B[a]P-4,5:7, 8-bis-diols. We conclude that trans-B[a]P-4,5-diol can be metabolized to a series of B[a]P-bis-diols, and can also be metabolically activated to form stable polar DNA adducts. The trans, trans-B[a]P-4,5:7,8-bis-diols were shown to be metabolic intermediates in the formation of these DNA adducts.

The comparative genotoxic effects of racemic trans-8,9-dihydroxy-8, 9-dihydrodibenzo[a,l]pyrene (trans-DB[a,l]P-8,9-diol), the metabolic K-region dihydrodiol of dibenzo[a,l] pyrene (DB[a,l]P) (dibenzo[def, p]chrysene) and DB[a,l]P in transformable mouse embryo C3H10T(1)/(2)Cl8 (C3H10T(1)/(2)) fibroblasts was investigated. The C3H10T(1)/(2) mouse embryo morphological cell-transforming activities of these polycyclic aromatic hydrocarbons (PAHs) were assayed using concentration-response studies. At concentrations of 33 nM and above both trans-DB[a,l]P-8,9-diol and DB[a,l]P produced significant (and similar) numbers of type II and III foci per dish and numbers of dishes with type II and II foci. Concomitant cytotoxicity studies revealed a reduction in colony survival of approximately 25% up to 198 nM for both PAHs. DNA adducts of trans-DB[a,l]P-8,9-diol and DB[a,l]P in C3H10T(1)/(2) cells were analyzed by a (32)P-post-labeling TLC/HPLC method. No adducts were observed in the DNA of C3H10T(1)/(2) cells treated with trans-DB[a, l]P-8,9-diol at concentrations that induced morphological cell transformation. Under the same exposure and chromatographic conditions, DNA adducts of deoxyadenosine and deoxyguanosine derived from the fjord region anti-DB[a,l]P-11,12-diol-13,14-epoxide and syn-DB[a,l]P-11,12-diol-13,14-epoxide were observed in the DNA of DB[a,l]P-treated cells. These results indicate that trans-DB[a,l]P-8, 9-diol has intrinsic genotoxic activity equal to that of DB[a,l]P, based on morphological cell transformation of mouse embryo fibroblasts. The activity of trans-DB[a,l]P-8,9-diol is apparently not associated with the formation of observable stable covalent DNA adducts. These results suggest that under appropriate conditions, trans-DB[a,l]P-8,9-diol may serve as an intermediate in the genotoxicity of DB[a,l]P.

This study examined the potential use of hemoglobin (Hb)- and serum-protein adducts of alachlor as potential biomarkers of alachlor exposure, a genotoxic and carcinogenic herbicide. The method developed was based on the observation that cleavage of S-cysteinyl alachlor-protein adducts by methanesulfonic acid gave the rearrangement product 3-(2',6'-diethylphenyl)-1, 3-thiazolidine-4-one (TZO). The structure of TZO was confirmed by mass spectroscopy, NMR spectroscopy, and independent synthesis. In the assay, treatment of alachlor-cysteinyl protein adducts by methanesulfonic acid was followed by extraction and analysis. TZO was detected and quantitated by electron-impact GC/MS in the single ion-monitoring mode. [ring-13C6]Alachlor-N-acetylcysteine was added as an internal standard prior to treatment and was converted to [ring-13C6]TZO, allowing response factors to be used to quantitate TZO concentrations. Incubations of alachlor (0-1000 microM) with human albumin and bovine serum albumin (BSA) resulted in linear adduct formation with both proteins. Maximal adduction levels of 613-1130 pmol alachlor-albumin adducts/mg protein were observed, with BSA binding close to twice that of human albumin. A linear concentration response of alachlor-Hb adducts was observed when whole blood from female CD rats was incubated with alachlor in vitro at concentrations up to 300 microM. Maximal binding was 1860 pmol alachlor-Hb adducts/mg globin. Male CD rats treated with alachlor at 150 mg/kg body wt/day ip for 0, 1, 2, and 3 days were sacrificed 4 days after final dosing. A maximal binding of 2250 pmol alachlor-Hb adducts/mg globin was observed. This assay provides a new approach for biomonitoring alachlor levels in experimental animals and has the potential for use in humans.

Adducts of the preemergence herbicide 2-chloro-N-(methoxymethyl)-N-(2,6-diethylphenyl)-acetamide (alachlor) and 2-chloro-N-(2,6-diethylphenyl)acetamide (CDEPA) with 2'-deoxyguanosine, thymidine, 2'-deoxyguanosine 3'-monophosphate, and thymidine 3'-monophosphate have been synthesized and characterized. Under mildly basic conditions alachlor and CDEPA form N-1 adducts with 2'-deoxyguanosine and N-3 adducts with thymidine as a result of chlorine displacement. In addition, alachlor formed an N-7 adduct with 2'-deoxyguanosine, 7-[[(N-(methoxymethyl)-N-(2,6-diethylphenyl)carbamoyl]methyl]guani ne. N-1 adducts of alachlor and CDEPA with 2'-deoxyguanosine 3'-monophosphate and N-3 adducts with thymidine 3'-monophosphate are also described. In addition to spectroscopic data, structural proof included the dephosphorylation of each nucleotide adduct to its corresponding nucleoside adduct by nuclease P1. Alachlor and alachlor adducts but not CDEPA and CDEPA adducts exhibited rotational isomerism as evidenced by proton and 13C NMR studies. These rotamers were attributed to hindered rotation about the shortened N-carbonyl bond. Computational methods employing molecular mechanics and quantum mechanics were used to characterize the structures and energies of these rotamers to account for the patterns of duplicate NMR resonances observed.

The ability of reduced polycyclic aromatic hydrocarbons to be converted to their fully aromatic forms by the microsomal cytochrome P-450 mixed-function oxidases may assist in the explanation of the mutagenic and tumorigenic activities of these agents. The metabolic conversion of 9,10-dihydrobenzo[a]pyrene (9,10-DHB[a]P) to benzo[a]pyrene (B[a]P) and 9- and/or 10-hydroxy-9,10-DHB[a]P (OH-9,10-DHB[a]P) was quantitatively measured. In beta-naphthoflavone-induced rat liver microsomes, 9,10-DHB[a]P was metabolized to B[a]P with a specific activity of 1.51 nmol B[a]P formed/min/mg microsomal protein. The formation of B[a]P was directly related to incubation time and microsomal protein concentration. Similarly, 9,10-DHB[a]P was converted to OH-9,10-DHB[a]P with a specific activity of 4.48 nmol OH-9,10-DHB[a]P formed/min/mg microsomal protein. Its formation was directly related to incubation time and microsomal protein concentration. The possibility of OH-9,10-DHB[a]P as a metabolic intermediate to B[a]P is discussed.