Retention of polybrominated diphenyl ethers and hydroxylated metabolites in paired human serum and milk in relation to CYP2B6 genotype.

Polybrominated diphenyl ethers (PBDEs) and their hydroxylated metabolites (OH-BDEs) are endocrine disrupting compounds prevalent in human serum and breast milk. Retention of PBDEs and OH-BDEs in humans may be affected by differences in PBDE metabolism due to variants in cytochrome P450 2B6 (CYP2B6). The objectives of this study are to assess the partitioning profiles of PBDEs and OH-BDEs in forty-eight paired human serum and milk samples, and to evaluate the relationship between variants in CYP2B6 genotype and PBDE and OH-BDE accumulation in humans. Results show that the geometric mean (GM) concentrations of PBDEs are similar in serum (GM = 43.4 ng/g lipid) and milk samples (GM = 52.9 ng/g lipid), while OH-BDEs are retained primarily in serum (GM = 2.31 ng/g lipid), compared to milk (GM = 0.045 ng/g lipid). Participants with CYP2B6*6 genotype had a greater relative retention of PBDEs in serum and milk, and significant relationships (p <  0.05) were also observed for PBDE-47, 5-OH-BDE-47 and 6-OH-BDE-47 concentrations relative to CYP2B6*5 and CYP2B6*6 genotypes. These results are the first to show that CYP2B6 genotype is significantly related to the relative retention of PBDEs in humans, which may have direct implications for variability in the susceptibility of individuals to the potential adverse effects of these contaminants.

Primary role of cytochrome P450 2B6 in the oxidative metabolism of 2,2',4,4',6-pentabromodiphenyl ether (BDE-100) to hydroxylated BDEs.

Human exposure to polybrominated diphenyl ethers (PBDEs) through various routes poses deleterious health effects. PBDEs are biotransformed into hydroxylated metabolites (OH-BDEs) via cytochrome P450s (P450s), which may add to their neurotoxic effects. This study characterizes the in vitro metabolism of 2,2',4,4',6-pentabromodiphenyl ether (BDE-100), one of the most abundant PBDE congeners found in humans, by recombinant human P450s and pooled human liver microsomes (HLMs). Ten recombinant P450s were individually incubated with BDE-100 to monitor P450-specific metabolism. P450 2B6 was found to be the predominant enzyme responsible for nearly all formation of six mono-OH-pentaBDE and two di-OH-pentaBDE metabolites. Four metabolites were identified as 3-hydroxy-2,2',4,4',6-pentabromodiphenyl ether (3-OH-BDE-100), 5'-hydroxy-2,2',4,4',6-pentabromodiphenyl ether (5'-OH-BDE-100), 6'-hydroxy-2,2',4,4',6-pentabromodiphenyl ether (6'-OH-BDE-100), and 4'-hydroxy-2,2',4,5',6-pentabromodiphenyl ether (4'-OH-BDE-103) through use of reference standards. The two remaining mono-OH-pentaBDE metabolites were hypothesized using mass spectral fragmentation characteristics of derivatized OH-BDEs, which allowed prediction of an ortho-OH-pentaBDE and a para-OH-pentaBDE positional isomer. Additional information based on theoretical boiling point calculations using COnductor-like Screening MOdel for Realistic Solvents (COSMO-RS) and experimental chromatographic retention times were used to identify the hypothesized metabolites as 2'-hydroxy-2,3',4,4',6-pentabromodiphenyl ether (2'-OH-BDE-119) and 4-hydroxy-2,2',4',5,6-pentabromodiphenyl ether (4-OH-BDE-91), respectively. Kinetic studies of BDE-100 metabolism using P450 2B6 and HLMs revealed Km values ranging from 4.9 to 7.0 µM and 6-10 µM, respectively, suggesting a high affinity toward the formation of OH-BDEs. Compared to the metabolism of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) reported in previous studies, BDE-100 appears to be more slowly metabolized by P450s due to the presence of a third ortho-substituted bromine atom.

Longitudinal assessment of occupational exposures to the organophosphorous insecticides chlorpyrifos and profenofos in Egyptian cotton field workers.

Chlorpyrifos (CPF) and profenofos (PFF) are organophosphorus (OP) insecticides that are applied seasonally in Egypt to cotton fields. Urinary trichloro-2-pyridinol (TCPy), a specific CPF metabolite, and 4-bromo-2-chlorophenol (BCP), a specific PFF metabolite, are biomarkers of exposure, while inhibition of blood butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) activities are effect biomarkers that may be associated with neurotoxicity. Urinary TCPy and BCP and blood BChE and AChE activities were measured in 37 adult Egyptian Ministry of Agriculture workers during and after 9-17 consecutive days of CPF application followed by an application of PFF (9-11 days), and a second CPF application (5 days) in 2008. During the OP applications, urinary TCPy and BCP levels were significantly higher than baseline levels, remained elevated following the application periods, and were associated with an exposure related inhibition of blood BChE and AChE. Analysis of blood AChE levels before and after the PFF application period suggests that individual workers with peak BCP levels greater than 1000 µg/g creatinine exhibited further inhibition of blood AChE with PFF application, demonstrating that PFF exposure had a negative impact on AChE activity in this highly exposed worker population. While large interindividual differences in exposure were observed throughout this longitudinal study (peak urinary BCP and peak TCPy levels for individuals ranging from 13.4 to 8052 and 16.4 to 30,107 µg/g creatinine, respectively), these urinary biomarkers were highly correlated within workers (r=0.75, p<0.001). This suggests that the relative exposures to CPF and PFF were highly correlated for a given worker. The variable exposures between job classification and work site suggest that job title and work location should not be used as the sole basis for categorizing OP exposures when assessing neurobehavioral and other health outcomes in Egyptian cotton field workers. Together, these findings will be important in educating the Egyptian insecticide application workers in order to encourage the development and implementation of work practices and personal protective equipment to reduce their exposure to CPF and PFF.

Metabolism of profenofos to 4-bromo-2-chlorophenol, a specific and sensitive exposure biomarker.

Profenofos is a direct acting phosphorothioate organophosphorus (OP) pesticide capable of inhibiting ß-esterases such as acetylcholinesterase, butyrylcholinesterase, and carboxylesterase. Profenofos is known to be detoxified to the biologically inactive metabolite, 4-bromo-2-chlorophenol (BCP); however, limited data are available regarding the use of urinary BCP as an exposure biomarker in humans. A pilot study conducted in Egyptian agriculture workers, demonstrated that urinary BCP levels prior to application (3.3-30.0 µg/g creatinine) were elevated to 34.5-3,566 µg/g creatinine during the time workers were applying profenofos to cotton fields. Subsequently, the in vitro enzymatic formation of BCP was examined using pooled human liver microsomes and recombinant human cytochrome P-450s (CYPs) incubated with profenofos. Of the nine human CYPs studied, only CYPs 3A4, 2B6, and 2C19 were able to metabolize profenofos to BCP. Kinetic studies indicated that CYP 2C19 has the lowest Km, 0.516 µM followed by 2B6 (Km=1.02 µM) and 3A4 (Km=18.9µM). The Vmax for BCP formation was 47.9, 25.1, and 19.2 nmol/min/nmol CYP for CYP2B6, 2C19, and 3A4, respectively. Intrinsic clearance (Vmax/Km) values of 48.8, 46.9, and 1.02 ml/min/nmol CYP 2C19, 2B6, and 3A4, respectively, indicate that CYP2C19 and CYP2B6 are primarily responsible for the detoxification of profenofos. These findings support the use of urinary BCP as a biomarker of exposure to profenofos in humans and suggest polymorphisms in CYP 2C19 and CYP 2B6 as potential biomarkers of susceptibility.

Biotransformation of BDE-47 to potentially toxic metabolites is predominantly mediated by human CYP2B6.

BACKGROUND: Previous studies have indicated that cytochrome P450s (CYPs) are involved in the metabolism of polybrominated diphenyl ether (PBDE) flame retardants in humans, resulting in the formation of hydroxylated PBDEs (OH-PBDEs) that are potentially more toxic than the parent PBDEs. However, the specific enzymes responsible for the formation of OH-PBDEs are unknown. OBJECTIVES: The purposes of this study were to characterize the in vitro metabolism of 2,2´,4,4´-tetrabromodiphenyl ether (BDE-47) by human liver microsomes (HLM) and recombinant human CYPs, and to identify the CYP(s) that are active in the oxidative metabolism of BDE-47. METHODS: Recombinant human CYPs (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) were incubated with BDE-47 (20 µM), and the metabolites were measured and characterized using gas chromatography with tandem mass spectrometry (GC-MS/MS). For kinetic studies, CYP2B6 and pooled human liver microsomes (HLMs) were incubated with BDE-47 (0-60 µM). RESULTS: CYP2B6 was the predominant CYP capable of forming six OH-BDEs, including 3-OH-BDE-47, 5-OH-BDE-47, 6-OH-BDE-47, 4-OH-BDE-42, 4´-OH-BDE-49, and a metabolite tentatively identified as 2´-OH-BDE-66. On the basis of full-scan GC-MS analysis, we hypothesized the formation of two other metabolites: di-OH-tetra-BDE and di-OH-tetrabrominated dioxin. In kinetic studies of BDE-47 metabolism by CYP2B6 and pooled HLMs, we found Km values ranging from 3.8 to 6.4 µM and 7.0 to 11.4 µM, respectively, indicating the high affinity toward the formation of OH-BDEs. CONCLUSION: Our findings support a predominant role of CYP2B6 in the metabolism of BDE-47 to potentially toxic metabolites, including a hypothesized di-OH-tetrabrominated dioxin metabolite. These results will assist future epidemiological studies investigating the potential of PBDEs and their metabolites to produce neurobehavioral/neurodevelopmental disorders.

Biomarkers of chlorpyrifos exposure and effect in Egyptian cotton field workers.

BACKGROUND: Chlorpyrifos (CPF), a widely used organophosphorus pesticide (OP), is metabolized to CPF-oxon, a potent cholinesterase (ChE) inhibitor, and trichloro-2-pyridinol (TCPy). Urinary TCPy is often used as a biomarker for CPF exposure, whereas blood ChE activity is considered an indicator of CPF toxicity. However, whether these biomarkers are dose related has not been studied extensively in populations with repeated daily OP exposures. OBJECTIVE: We sought to determine the relationship between blood ChE and urinary TCPy during repeated occupational exposures to CPF. METHODS: Daily urine samples and weekly blood samples were collected from pesticide workers (n=38) in Menoufia Governorate, Egypt, before, during, and after 9-17 consecutive days of CPF application to cotton fields. We compared blood butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) activities with the respective urinary TCPy concentrations in each worker. RESULTS: Average TCPy levels during the middle of a 1- to 2-week CPF application period were significantly higher in pesticide applicators (6,437 µg/g creatinine) than in technicians (184 µg/g) and engineers (157 µg/g), both of whom are involved in supervising the application process. We observed a statistically significant inverse correlation between urinary TCPy and blood BuChE and AChE activities. The no-effect level (or inflection point) of the exposure-effect relationships has an average urinary TCPy level of 114 µg/g creatinine for BuChE and 3,161 µg/g creatinine for AChE. CONCLUSIONS: Our findings demonstrate a dose-effect relationship between urinary TCPy and both plasma BuChE and red blood cell AChE in humans exposed occupationally to CPF. These findings will contribute to future risk assessment efforts for CPF exposure.

Analysis of hydroxylated polybrominated diphenyl ether metabolites by liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry.

Hydroxylated polybrominated diphenyl ether (OH-PBDEs) metabolites have the potential to cause endocrine disruption as well as other health effects. Currently, gas chromatography/mass spectrometry (GC/MS) after derivatization is used for the analysis of OH-PBDEs. However, there is a need for the direct analysis of OH-PBDEs at relatively low concentrations in environmental and biological samples. Liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry (LC/APCI-MS/MS) was evaluated for the analysis of nine OH-PBDEs, ranging from tri- to hexabrominated. Separation of the nine isomeric metabolites was achieved with reversed-phase liquid chromatography, followed by detection by APCI-MS in negative mode. Notably, a significant decrease in ionization was observed in 6-hydroxyl-substituted PBDE metabolites in the presence of an ortho-substituted bromine, relative to the other hydroxylated metabolites. This is probably due to the formation of dioxins in the source as a result of the high-temperature conditions, which prevented ionization by hydrogen abstraction. The MS/MS experiments also provided evidence of the neutral losses of HBr and Br(2), indicating the possible use of neutral loss scanning and selected reaction monitoring (SRM) for the screening of brominated metabolites in samples. The applicability of LC/APCI-MS/MS was demonstrated for the analysis of metabolites of BDEs 47 and 99 formed in human liver microsomes. The LC/APCI-MS/MS method was able to detect metabolites that had previously been identified by GC/MS following derivatization.

Human liver microsome-mediated metabolism of brominated diphenyl ethers 47, 99, and 153 and identification of their major metabolites.

While the metabolism and excretion of polybrominated diphenyl ethers (PBDEs) have been reported in rodents, PBDE metabolism in humans has only recently been investigated. In this present study, individual human liver microsomes were incubated for 120 min with radiolabeled and nonradiolabeled BDE 47, 99, or 153 to determine their relative degrees of metabolism and to identify the structures of metabolites formed. Radiolabeled samples were analyzed using high-performance liquid chromatography/radiochemical detection, while nonradiolabeled samples were analyzed with and without derivatization using gas chromatography/mass spectrometry. Results from radiolabeled incubations demonstrated that human liver microsomes metabolized BDEs 47 and 99 but not BDE 153. Differences in the extent of BDE metabolism by the three individual liver specimens used in the study were observed. BDE 47 metabolized to a dihydroxylated BDE 47 and 2,4-dibromophenol, while BDE 99 metabolized to a dihydroxylated BDE 99, 2,4,5-tribromophenol and 1,3-dibromobenzene. This study showed that BDEs 47 and 99 are metabolized by human liver microsomes with relatively large interindividual differences. Results of this study could provide one explanation for the high bioaccumulation rate of BDE 153 in humans.

Lung development in the Holtzman rat is adversely affected by gestational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant that elicits a wide range of toxic effects on the developing organism. In this study, we demonstrate that the fetal and neonatal rat lung contains a responsive Ahr-signaling pathway which upon activation by a gestational exposure to TCDD, leads to altered lung development. Pregnant Holtzman rats received a single oral dose of TCDD (1.5 or 6 microg/kg) on gestation day (GD) 10 or a vehicle control with fetal and neonatal analysis occurring on GD20 or postnatal day (PND) 7. Components of the aryl hydrocarbon receptor (Ahr) signaling pathway (Ahr and Arnt) were identified in the fetal and neonatal lung tissue through the use of real-time PCR and immunohistochemical staining at both time points. Additionally, the Ahr-signaling pathway was found to be responsive to the gestational TCDD exposure as demonstrated by the induction of Cyp1a1, Cyp1b1, and Ahrr in both fetal and neonatal lung tissue. Morphometric analysis of GD20 and PND7 fixed lung tissue sections revealed that treated pups had significant decreases in total airspace area while having significantly wider tissue septa separating the airspaces as well as a decreased dry lung weight to body weight ratio when compared with controls; indicative of lung immaturity and hypoplasia. Finally, the assessment of respiratory mechanics on PND7 pups revealed functionally different pressure-volume curves in TCDD-exposed pups when compared with control animals. Together, these data identify a responsive Ahr-signaling pathway in the developing lung which may be related to the pulmonary immaturity and hypoplasia induced by TCDD and demonstrates that gestational exposure to TCDD alters lung development in such a manner that changes in lung morphology are associated with functional differences in respiratory mechanics.

Effects of Helicobacter infection on developmental toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in Holtzman rats.

To obtain accurate results from experiments in animal models, all potential confounding variables must be identified and controlled. The authors examined the effects of Helicobacter infection on developmental toxicity resulting from exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a persistent and ubiquitous environmental contaminant. They administered different doses of TCDD to timed pregnant Helicobacter-positive and Helicobacter-negative Holtzman rats and evaluated fetal and neonatal viability. They also assessed hepatic cytochrome P450 induction and activity of the gene Cyp1a1, which are classic indicators of TCDD exposure. All rats were affected by TCDD, and Helicobacter infection seemed to have little influence on rats' susceptibility to the compound.

Gestational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin alters retinoid homeostasis in maternal and perinatal tissues of the Holtzman rat.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), one of the most widely studied environmental contaminants, causes a variety of adverse health effects including teratogenesis and altered development which may be related to disruptions in retinoid homeostasis. The purpose of this study was to determine the effect that gestational administration of TCDD has on retinoid homeostasis in both pregnant Holtzman rats and developing fetuses and neonates. A single oral dose of TCDD (0, 1.5, 3, or 6 microg/kg) was administered to pregnant rats on gestation day 10, with fetuses analyzed on gestation days 17 and 20, and neonates analyzed on post natal day 7. Exposure to TCDD generally produced decreases in the concentrations of retinyl esters, such as retinyl palmitate, and retinol in maternal and perinatal liver and lung, while increasing levels in the maternal kidney. Additionally, perinatal hepatic retinol binding protein 1-dependent retinyl ester hydrolysis was also decrease by TCDD. Sensitivity of the developing perinates to TCDD appeared to have an age-related component demonstrated by an increased rate of mortality and significant alterations to body weight and length on post natal day 7 relative to that observed at gestation day 20. A unique observation made in this study was a significant decrease in lung weight observed in the perinates exposed to TCDD. Taken together, these data demonstrate that TCDD significantly alters retinoid homeostasis in tissues of the developing fetus and neonate, suggesting that their unique sensitivity to TCDD may at least be in part the result of altered retinoid homeostasis.

Induction of CYP2B and CYP2E1 in precision-cut rat liver slices cultured in defined medium.

Many drugs and endogenous substances undergo biotransformation by cytochrome P450s (CYPs), and some drugs are also capable of modulating the expression of various CYPs. Knowledge of the potential of a drug to modulate CYPs is useful to help predict potential drug interactions. This study utilized precision-cut rat liver slices in dynamic organ culture to assess the effects of various media on the viability of rat liver slices and the expression of CYP2B and CYP2E1 when the slices are exposed to phenobarbital and isoniazid, which are drugs capable of inducing these respective CYPs. Liver slices were maintained in serum supplemented Waymouths medium and two different serum-free media, Hepatozyme (Life Technologies) and a new defined medium, which is named BPM. While Hepatozyme is considered a suitable medium to support primary hepatocyte cultures, this product did not maintain viable liver slices, even for 24 h. The serum containing and new defined media maintained viable liver slices for up to 96 h in culture. Phenobarbital (0.5 mM) and isoniazid (0.1 or 0.6 mM) did not affect viability in this model. In the absence of phenobarbital or isoniazid, liver slices maintained for 96 h in the new BPM medium maintained the respective levels of CYP2B and 2E1 protein at 1.8 and 1.9-fold higher than in slices maintained in the serum-containing medium. Phenobarbital exposure (0.5 mM) for 96 h induced CYP2B protein 5.2-fold in the BPM medium and 2.5-fold in the serum-containing medium. Isoniazid exposure (0.1 and 0.5 mM) for 96 h induced CYP2E1 protein 1.9 and 2.1-fold (respectively) in the BPM medium and 2.1 and 2.0-fold in the serum-containing medium. The respective CYP enzymatic activities were also increased by these drugs in a similar manner. Thus, the new defined BPM medium provides suitable conditions for maintaining CYP2B and 2E1 in liver slices and supports the investigation of drug-induced modulation of these enzymes.

Human hepatic cytochrome p450-specific metabolism of parathion and chlorpyrifos.

Organophosphorus pesticides (OPs) remain a potential concern to human health because of their continuing worldwide use. Thiophosphorus OPs, once bioactivated by cytochromes P450 (P450s), form oxon metabolites, which are potent acetylcholinesterase inhibitors. This study investigated the rate of desulfation (activation) and dearylation (detoxification) of parathion and chlorpyrifos in human liver microsomes. In addition, recombinant human P450s were used to quantify, for the first time, the P450-specific kinetic variables (K(m) and V(max)) for each compound for future use in refining human physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) models of OP exposure. CYP1A2, 2B6, 2C9, 2C19, 3A4, 3A5, and 3A7 were found to be active to a widely varying degree in parathion metabolism, whereas all, with the exception of CYP2C9, were also found to be active in chlorpyrifos metabolism. CYP2B6 and CYP2C19 demonstrated low K(m) and high V(max) values for the metabolism of both model compounds, which supports their role as the primary enzymes that regulate metabolism at low-level human exposures to OPs. With K(m) and V(max) values of 0.61 microM, 4827 pmol/min/nmol P450 and 0.81 microM, 12,544 pmol/min/nmol for formation of paraoxon and chlorpyrifos-oxon, respectively, CYP2B6 favored the desulfation reaction. CYP2C19 activity favored dearylation with K(m) and V(max) values of 0.60 microM, 2338 pmol/min/nmol P450 and 1.63 microM, 13,128 pmol/min/nmol for formation of p-nitrophenol and 3,4,5-tricholorpyrindinol, respectively. P450-specific kinetic parameters for OP metabolism will be used with age-dependent hepatic P450 content to enhance PBPK/PD models so that OP exposures can be modeled to protect human health in different age groups.

Comparative developmental toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the hamster, rat and guinea pig.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant capable of causing a wide variety of adverse health effects including teratogenesis and altered development. The objective of this study was to compare the developmental toxicity of TCDD in the hamster, rat and guinea pig, which in mature animals exhibit a relatively low, medium and high sensitivity to TCDD, respectively. A single oral dose of TCDD was administered to pregnant rats (0, 1.5, 3.0, 6.0 or 18.0microg/kg) on gestation day 10, pregnant hamsters (0, 1.5, 3.0, 6.0 or 18.0microg/kg) on gestation day 9 and pregnant guinea pigs (0, 0.15 or 1.5microg/kg) on gestation day 14 with fetal analysis on gestation day 20, 15 and 56, respectively. The developmental toxicity of TCDD in the three species included increased fetal mortality, alterations to fetal body weight, body length, organ weight and significant changes to the fetal white blood cell differential counts. Additionally, teratogenic responses were observed in the hamster and rat consisting of cleft palate, kidney congestion, hydronephrosis and intestinal hemorrhaging. Furthermore, the results from this study demonstrate that despite the up to 5000-fold interspecies variability to the acute lethal potency of TCDD observed in mature guinea pigs, rats and hamsters, the developing fetus is uniquely vulnerable to gestational TCDD exposure and displays approximately a 10-fold variability in fetal lethal potency in these species. Together, these results will assist efforts to reduce the uncertainty in the risk assessment for TCDD in sensitive populations, such as the developing embryo and fetus.

Hepatic gene downregulation following acute and subchronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to lead to the development of hepatotoxicity and carcinogenicity in the liver of female rats. In this study, we investigated hepatic gene downregulation in response to acute and subchronic TCDD exposure. We identified 61 probes which exhibited a downregulation of twofold or greater following subchronic (13 weeks) exposure to TCDD. Comparative analysis of the hepatic expression of these 61 probes was conducted with rats subchronically exposed to PeCDF, PCB126, PCB153, and a mixture of PCB126 and PCB153. PCB153 produced little or no alteration in these probes, while the binary mixture mimicked most closely the downregulation observed with TCDD. To discern if the repression of genes within this probe set occur as a primary response to TCDD exposure, we analyzed the early responsiveness of 11 genes at 6, 24, and 72 h following a single exposure to TCDD. We observed early repression of the 11 genes within this early time course, indicating that the repression of this subset of genes occurs as a primary response to TCDD exposure and not as a secondary response to 13 weeks of subchronic treatment. In addition, the gender, species, and AhR dependence of these responses were also investigated. Gender- and species-dependent repression was observed within this subset of genes. Furthermore, utilizing AhR knockout mice, we were able to determine the AhR-dependent downregulation of seven of 11 genes. Together these results assist efforts to understand the multitude of effects imposed by TCDD and AhR ligands on gene expression.

Tissue specific induction of cytochrome P450 (CYP) 1A1 and 1B1 in rat liver and lung following in vitro (tissue slice) and in vivo exposure to benzo(a)pyrene.

Cytochrome P-450s (CYPs) detoxify a wide variety of xenobiotics and environmental contaminants, but can also bioactivate carcinogenic polycyclic aromatic hydrocarbons, such as benzo(a)pyrene (BaP), to DNA-reactive species. The primary CYPs involved in the metabolism and bioactivation of BaP are CYP1A1 and CYP1B1. Furthermore, BaP can induce expression of CYP1A1 and CYP1B1 via the aryl hydrocarbon receptor. Induction of CYP1A1 and CYP1B1 by BaP in target (lung) and non-target (liver) tissues was investigated utilizing precision-cut rat liver and lung slices exposed to BaP in vitro. Tissue slices were also prepared from rats pretreated in vivo with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to induce expression of CYP1A1 and CYP1B1. In addition, in vivo exposure studies were performed with BaP to characterize and validate the use of the in vitro tissue slice model. In vitro exposure of liver and lung slices to BaP resulted in a concentration-dependent increase in CYP1A1 and CYP1B1 mRNA and protein levels, which correlated directly with the exposure-related increase in BaP-DNA adduct levels observed previously in the tissue slices [Harrigan, J.A., Vezina, C.M., McGarrigle, B.P., Ersing, N., Box, H.C., Maccubbin, A.E., Olson, J.R., 2004. DNA adduct formation in precision-cut rat liver and lung slices exposed to benzo(a)pyrene. Toxicological Sciences 77, 307-314]. Pretreatment of animals in vivo with TCDD produced a marked induction of CYP1A1 and CYP1B1 expression in the tissue slices, which was similar to the levels of CYP1A1 and CYP1B1 mRNA achieved in liver and lung following in vivo treatment with BaP. Following in vitro exposure to BaP, the levels of CYP1A1 were greater in the lung than the liver, while following all exposures (in vitro and in vivo), the levels of CYP1B1 mRNA were greater in lung tissue compared to liver. The higher expression of CYP1A1 and CYP1B1 in the lung was associated with higher levels of BaP-DNA adducts in the lung slices (Harrigan et al.'s work) and together, these results may contribute to the tissue specificity of BaP-mediated carcinogenesis.

Formation of tamoxifen-DNA adducts in human endometrial explants exposed to alpha-hydroxytamoxifen.

An increased risk of developing endometrial cancer has been observed in women receiving tamoxifen (TAM) endocrine therapy and chemoprevention. The genotoxic damage induced by TAM metabolites may be involved in the development of endometrial cancer. To investigate the capability of endometrial tissues to form TAM-DNA adducts, primary cultured human endometrial explants were exposed to alpha-hydroxytamoxifen (alpha-OHTAM) and used for quantitative analysis of TAM-DNA adducts, using (32)P-postlabeling/HPLC analysis. A trans isoform of alpha-(N(2)-deoxyguanosinyl)tamoxifen (dG-N(2)-TAM) was detected as the major adduct in eight of nine endometrial explants exposed to 100 microM alpha-OHTAM at levels of 7.7 +/- 5.3 (mean +/- SD) adducts/10(7) nucleotides. Approximately 25- and 37-fold lower amounts of the cis form of dG-N(2)-TAM and another trans isoform were also detected. The dG-N(2)-TAM adduct (3.3 adducts/10(7) nucleotides) was detected in one of three endometrial explants exposed to 25 microM alpha-OHTAM. No TAM-DNA adducts were detected in any unexposed tissues. These results indicate that TAM-DNA adducts are capable of forming through O-sulfonation and/or O-acetylation of alpha-OHTAM in the endometrium. The endometrial explant culture can be used as a model system to explore the genotoxic mechanism of antiestrogens for humans.

DNA adduct formation in precision-cut rat liver and lung slices exposed to benzo[a]pyrene.

Chemical-DNA adducts provide an integrated measure of exposure, absorption, bioactivation, detoxification, and DNA repair following exposure to a genotoxic agent. Benzo[a]pyrene (BaP), a prototypical polycyclic aromatic hydrocarbon (PAH), can be bioactivated by cytochrome P-450s (CYPs) and epoxide hydrolase to genotoxic metabolites which form covalent adducts with DNA. In this study, we utilized precision-cut rat liver and lung slices exposed to BaP to investigate tissue-specific differences in chemical absorption and formation of DNA adducts. To investigate the contribution of bioactivating CYPs (such as CYP1A1 and CYP1B1) on the formation of BaP-DNA adducts, animals were also pretreated in vivo with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) prior to in vitro incubation of tissue slices with BaP. Furthermore, the tissue distribution of BaP and BaP-DNA adduct levels from in vivo studies were compared with those from the in vitro tissue slice experiments. The results indicate a time- and concentration-dependent increase in tissue-associated BaP following exposure of rat liver and lung tissue slices to BaP in vitro, with generally higher levels of BaP retained in lung tissue. Furthermore, rat liver and lung slices metabolized BaP to reactive intermediates that formed covalent adducts with DNA. Total BaP-DNA adducts increased with concentration and incubation time. Adduct levels (fmol adduct/microg DNA) in lung slices were greater than liver at all doses. Liver slices contained one major and two minor adducts, while lung slices contained two major and 3 minor adducts. The tissue-specific qualitative profile of these adducts in tissue slices was similar to that observed from in vivo studies, further validating the use of this model. Pretreatment of animals with TCDD prior to in vitro incubation with BaP potentiated the levels of DNA adduct formation. TCDD pretreatment altered the adduct distribution in lung but not in liver slices. Together, the results suggest that tissue-specific qualitative and quantitative differences in BaP-DNA adducts could contribute to the lung being a target tissue for BaP carcinogenesis. Furthermore, the results validate the use of precision-cut tissue slices incubated in dynamic organ culture as a useful model for the study of chemical-DNA adduct formation.

Biotransformation of tamoxifen in a human endometrial explant culture model.

Although long-term tamoxifen therapy is associated with increased risk of endometrial cancer, little is known about the ability of endometrial tissue to biotransform tamoxifen to potentially reactive intermediates, capable of forming DNA adducts. The present study examined whether explant cultures of human endometrium provide a suitable in vitro model to investigate the tissue-specific biotransformation of tamoxifen. Fresh human endometrial tissue, microscopically uninvolved in disease, was cut into 1 x 2-mm uniform explants and incubated with media containing either 25 or 100 microM tamoxifen in a 24-well plate. Metabolites were analyzed by reversed-phase HPLC using postcolumn, online, photochemical activation and fluorescence detection. Three metabolites, namely, alpha-hydroxytamoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen were identified in culture medium and tissue lysates. N-desmethyltamoxifen was found to be the major metabolite in both tissue and media extracts of tamoxifen-exposed explants. Incubations of tamoxifen with recombinant human cytochrome P-450s (CYPs) found that CYP2C9 and CYP2D6 produced all three of the above tamoxifen metabolites, while CYP1A1 and CYP3A4 catalyzed the formation of alpha-hydroxytamoxifen and N-desmethyltamoxifen, and CYP1A2 and CYP1B1 only formed the alpha-hydroxy metabolite. CYP2D6 exhibited the greatest activity for the formation of all three tamoxifen metabolites. Western immunoblots of microsomes from human endometrium detected the presence of CYPs 2C9, 3A, 1A1 and 1B1 in fresh endometrium, while CYPs 2D6 and 1A2 were not detected. Immunohistochemical (IHC) analysis also confirmed the presence of CYPs 2C9, 3A and 1B1 in fresh human endometrium and in viable tissue cultured for 24 h with or without tamoxifen. Together, the results support the use of explant cultures of human endometrium as a suitable in vitro model to investigate the biotransformation of tamoxifen in this target tissue. In addition, the results support the role of CYPs 2C9, 3A, 1A1 and 1B1 in the biotransformation of tamoxifen, including the formation of the DNA reactive alpha-hydroxytamoxifen metabolite, in human endometrium.

Antioxidant inhibits tamoxifen-DNA adducts in endometrial explant culture.

Fresh human endometrial explants were incubated for 24h at 37 degrees C with either tamoxifen (10-100 micro M) or the vehicle (0.1% ethanol). Three metabolites namely, alpha-hydroxytamoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen were identified in the culture media. Tissue size was limited but DNA adducts formed by the alpha-hydroxytamoxifen pathway were detected using authentic alpha-(deoxyguanosyl-N(2)) tamoxifen standards. Relative DNA-adduct levels of 2.45, 1.12, and 0.44 per 10(6) nucleotides were detected following incubations with 100, 25, and 10 micro M tamoxifen, respectively. The concurrent exposure of the explants to 100 micro M tamoxifen with 1mM ascorbic acid reduced the level of alpha-hydroxytamoxifen substantially (68.9%). The formation of tamoxifen-DNA adducts detectable in the explants from the same specimens exposed to 100 micro M tamoxifen with 1mM ascorbic acid were also inhibited. These results support the role of oxidative biotransformation of tamoxifen in the subsequent formation of DNA adducts in this tissue.