Comparative cytotoxic effects of acetaminophen (N-acetyl-p-aminophenol), a non-hepatotoxic regioisomer acetyl-m-aminophenol and their postulated reactive hydroquinone and quinone metabolites in monolayer cultures of mouse hepatocytes

Toxic effects of acetaminophen (paracetamol, N-acetyl-p-aminophenol, APAP) in monolayer cultures of mouse hepatocytes developed over a period of 18 hr. N-Acetyl-m-aminophenol (AMAP) was approximately 10-fold less toxic than APAP, despite the fact that it bound covalently to a greater extent to hepatocyte macromolecules. AMAP did not deplete glutathione to as great an extent as APAP, indicating that their reactive metabolites may bind to different proteins or that oxidative damage in addition to arylation of proteins may be involved in the development of cell death. The toxicity of 3-methoxy-acetyl-p-aminophenol was similar to that of APAP, whereas the other hydroquinone and quinone metabolites were 8-10 times more cytotoxic than APAP. The potencies of these analogs were in the order: acetyl-m-aminophenol-p-benzoquinoneimine greater than or equal to 2,5-dihydroxyacetanilide greater than or equal to 3-methoxy-p-benzoquinone greater than or equal to N-acetyl-p-benzoquinone imine (NAPQI) greater than or equal to acetyl-m-aminophenol-o-benzoquinone greater than or equal to 3-hydroxy-acetyl-p-aminophenol. The relative toxic potencies of the hydroquinone and quinone metabolites of AMAP were comparable to that of NAPQI, and do not readily explain the marked difference between the cytotoxic effects of AMAP and APAP.     

Sulfhydryl binding and topoisomerase inhibition by PCB metabolites

Polychlorinated biphenyls (PCBs) are highly persistent contaminants in our environment. Their persistence is due to a general resistance to metabolic attack. Lower halogenated PCBs, however, are metabolized to mono- and dihydroxy compounds, and the latter may be further oxidized to quinones with the formation of reactive oxygen species (ROS). We have shown that PCB metabolism generates ROS in vitro and in cells in culture and this leads to oxidative DNA damage, like DNA strand breaks and 8-oxo-dG formation. In the present study, we have evaluated the reactivity of PCB metabolites with other nucleophiles, like glutathione (GSH), by assessing (1) quantitative GSH binding in vitro, (2) GSH and thiol (sulfhydryl) depletion in HL-60 cells, (3) the associated cytotoxicity, and (4) the inhibition of topoisomerase II activity in vitro. PCB quinones were found to bind GSH in vitro at a ratio of 1:1.5 and to deplete GSH in HL-60 cells as measured by both spectrophotometric and spectrofluorometric methods. By flow cytometry analysis, we confirmed that there was intracellular GSH depletion in HL-60 cells by PCB quinones and this is associated with cytotoxicity. On the other hand, the PCB hydroquinone metabolites did not bind GSH or other thiols within 1 h of exposure. However, by spectral analyses we found that the PCB hydroquinones could be oxidized enzymatically to the quinones, which could then bind GSH. The resulting hydroquinone-glutathione addition product(s) could undergo a second and third cycle of oxidation and GSH addition with the formation of di- and tri-GSH-PCB adducts. The effect of the PCB metabolites was also tested on a sulfhydryl-containing enzyme, topoisomerase II. PCB quinones inhibited topoisomerase II activity while the PCB hydroquinone metabolites did not. Hence, the oxidation of PCB hydroquinone metabolites to quinones in cells followed by the binding of quinones to GSH and to protein sulfhydryl groups and the resulting oxidative stress may be important aspects of the toxicity of these compounds.     

Semiquinone radicals from oxygenated polychlorinated biphenyls: electron paramagnetic resonance studies

Polychlorinated biphenyls (PCBs) can be oxygenated to form very reactive hydroquinone and quinone products. A guiding hypothesis in the PCB research community is that some of the detrimental health effects of some PCBs are a consequence of these oxygenated forms undergoing one-electron oxidation or reduction, generating semiquinone radicals (SQ (*-)). These radicals can enter into a futile redox cycle resulting in the formation of reactive oxygen species, that is, superoxide and hydrogen peroxide. Here, we examine some of the properties and chemistry of these semiquinone free radicals. Using electron paramagnetic resonance (EPR) to detect SQ (*-) formation, we observed that (i) xanthine oxidase can reduce quinone PCBs to the corresponding SQ (*-); (ii) the heme-containing peroxidases (horseradish and lactoperoxidase) can oxidize hydroquinone PCBs to the corresponding SQ (*-); (iii) tyrosinase acting on PCB ortho-hydroquinones leads to the formation of SQ (*-); (iv) mixtures of PCB quinone and hydroquinone form SQ (*-) via a comproportionation reaction; (v) SQ (*-) are formed when hydroquinone-PCBs undergo autoxidation in high pH buffer (approximately >pH 8); and, surprisingly, (vi) quinone-PCBs in high pH buffer can also form SQ (*-); (vii) these observations along with EPR suggest that hydroxide anion can add to the quinone ring; (viii) H 2 O 2 in basic solution reacts rapidly with PCB-quinones; and (ix) at near-neutral pH SOD can catalyze the oxidization of PCB-hydroquinone to quinone, yielding H 2 O 2. However, using 5,5-dimethylpyrroline-1-oxide (DMPO) as a spin-trapping agent, we did not trap superoxide, indicating that generation of superoxide from SQ (*-) is not kinetically favorable. These observations demonstrate multiple routes for the formation of SQ (*-) from PCB-quinones and hydroquinones. Our data also point to futile redox cycling as being one mechanism by which oxygenated PCBs can lead to the formation of reactive oxygen species, but this is most efficient in the presence of SOD.     

Production of DNA strand breaks in vitro and reactive oxygen species in vitro and in HL-60 cells by PCB metabolites.

PCBs are industrial chemicals that continue to contaminate our environment. They cause various toxic effects in animals and in exposed human populations. The mechanisms of toxicity, however, are not completely understood. PCBs are metabolized by cytochromes P450 to mono- and dihydroxylated compounds. Dihydroxy-PCBs can potentially be oxidized to the corresponding quinones. We hypothesized that reactive oxygen species (ROS) are produced by redox reactions of PCB metabolites. We tested several synthetic dihydroxy- and quinoid-PCBs with 1-3 chlorines for their potential to produce ROS in vitro and in HL-60 human leukemia cells, and DNA strand breaks in vitro. All dihydroxy-PCBs tested produced superoxide. The quinones generated superoxide only in the presence of GSH, probably during the autoxidation of the glutathione conjugates. We observed increased superoxide production with decreasing halogenation. Incubation of dihydroxy-PCBs or PCB quinones + GSH with plasmid DNA resulted in DNA strand break induction in the presence of Cu(II). Tests with various ROS scavengers indicated that hydroxyl radicals and singlet oxygen are likely involved in this strand break induction. Finally, dihydroxy- and quinoid PCBs also produced ROS in HL-60 cells in a dose- and time-dependent manner. We conclude that dihydroxylated PCBs, and PCB quinones after reaction with GSH, produce superoxide and other ROS both in vitro and in HL-60 cells, and oxidative DNA damage in the form of DNA strand breaks in vitro. The reactions seen in vitro and in cells may well be a predictor of the toxicity of PCBs in animals.     

Cytotoxic effects of phenyl-hydroquinone and some hydroquinones on isolated rat hepatocytes.

The cytotoxic effects of phenyl-hydroquinone (PHQ) and some other hydroquinones on freshly isolated rat hepatocytes were investigated. Addition of PHQ (0.5 or 0.75 mM) to the hepatocytes elicited dose-dependent cell death accompanied by losses of intracellular glutathione (GSH), protein thiols and ATP. These effects were related to both PHQ loss and phenyl-benzoquinone (PBQ) formation in the cell suspension. The cytotoxicity of PHQ was prevented by sulphydryl compounds such as cysteine and GSH. In Krebs-Henseleit buffer without cells, loss of PHQ (0.5 mM; initial concentration) and formation of PBQ, monitored by spectral measurements, were inhibited by addition of 50 microM GSH. Further, the oxygen consumption owing to autoxidation of PHQ (0.5 mM) in Krebs-Henseleit buffer without cells was depressed by addition of 50 microM GSH. Among all the hydroquinones tested (at 0.5 mM), tert-butyl-hydroquinone and PHQ were most toxic, followed by hydroquinone and 2,5-di(tert-butyl)-1,4-benzohydroquinone. However, accumulation of cellular malondialdehyde was not affected by these hydroquinones. The toxicity was related to the rate of oxygen consumption by each hydroquinone in the buffer. These results suggest that hydroquinone-induced cytotoxicity is dependent on the rate of oxidation of these compounds as well as the loss of protein thiols.     

Benzene's toxicity: a consolidated short review of human and animal studies by HA Khan

Khan's review is a brief summary of the complex field of study revolving around bone marrow toxicity and leukemogenesis observed in people chronically exposed to benzene. These comments are intended to demonstrate the use of the Kahn review as a launching pad for an in-depth analysis of the several related areas that must be fully explored to understand benzene-related diseases. The accumulated evidence demonstrates that benzene-induced bone marrow damage results from the production of hematotoxins that are metabolic products of benzene metabolism. The metabolism of benzene is described with respect to the formation benzene metabolites with emphasis on phenol and hydroquinone, which are the major metabolites, the significance of the formation of glutathione conjugates, the activity of NAD(P)H:quinone oxidoreductase (NQO1), and the ring opening products. Results are shown suggesting that oxidative stress induced by benzene metabolites is likely to be a significant factor in damaging DNA in bone marrow cells. Although a variety of effects on bone marrow can be demonstrated it is not yet clear which metabolites are most important in either benzene-induced aplastic anemia or leukemia. Benzene metabolism alone is insufficient to fully describe benzene toxicity. The impact of benzene metabolites on bone marrow cells must be fully explored to determine how benzene exposure can result in decreased viability or genetic toxicity to cells in the bone marrow.     

Mechanisms of toxic injury to isolated hepatocytes by 1-naphthol

The mechanism(s) of toxicity of 1-naphthol and two of its possible metabolites, 1,2- and 1,4-naphthoquinone, to freshly isolated rat hepatocytes has been studied. 1-Naphthol and both naphthoquinones exhibited a dose-dependent toxicity to hepatocytes. [1-14C]-1-Naphthol was metabolised by hepatocytes predominantly to its glucuronic acid and sulphate ester conjugates, but small amounts of covalently bound products were also formed. Blebbing on the surface of the hepatocytes was observed following exposure to 1-naphthol and the naphthoquinones, together with a dose-dependent decrease in intracellular glutathione (GSH), which preceded the onset of cytotoxicity. The toxicity of 1-naphthol and the naphthoquinones was potentiated by dicoumarol, an inhibitor of DT-diaphorase (NAD(P)H:quinone oxidoreductase). This enhanced toxicity was accompanied by a greater amount of surface blebbing, an increased depletion of intracellular GSH, particularly in the case of 1-naphthol and 1,4-naphthoquinone, and a decreased metabolism of 1-naphthol to its conjugates with variable effects on the amount of covalently bound products formed. These results support the suggestion that the toxicity of 1-naphthol may be mediated by the formation of 1,2-naphthoquinone and/or 1,4-naphthoquinone, which may then be metabolised by one electron reduction to naphthosemiquinone radicals. These, in turn, may covalently bind to important cellular macromolecules or enter a redox cycle with molecular oxygen thereby generating active oxygen species. Both of these processes appear to play a role in producing the cytotoxic effects of 1-naphthol.     

Studies on the mechanism of coumarin-induced toxicity in rat hepatocytes: comparison with dihydrocoumarin and other coumarin metabolites

Single doses of coumarin (125 mg/kg, ip) produced a depletion of hepatic nonprotein sulfhydryl groups (mainly reduced glutathione; GSH) in young male Sprague-Dawley rats after 2 hr and increased liver weight and produced hepatic centrilobular necrosis after 24 hr. Coumarin also produced time- and dose-dependent toxic effects in primary rat hepatocyte cultures. A marked reduction of GSH levels was also observed in vitro and this was not due either to the formation of oxidized glutathione (GSSG) or to the leakage of GSH and/or GSSG from the hepatocytes. Coumarin-induced toxicity in rat hepatocytes could be inhibited by the cytochrome P450 inhibitors ellipticine and metyrapone and potentiated by depleting hepatocyte GSH levels with diethyl maleate. In contrast to coumarin, dihydrocoumarin--which lacks the 3,4-double bond--produced little toxicity in rat hepatocytes either in vivo (127 and 254 mg/kg, ip) or in vitro. Similarly, coumarin was more toxic to rat hepatocytes than a number of known coumarin metabolites including 3- and 7-hydroxycoumarin and o-hydroxyphenylacetic acid. The results of these studies demonstrate a good in vivo/in vitro correlation for the effects of coumarin and dihydrocoumarin in rat hepatocytes. Furthermore, the data suggest that coumarin hepatoxicity in the rat is due to coumarin bioactivation by cytochrome P450-dependent enzymes to a toxic metabolite(s), which may be a coumarin 3,4-epoxide intermediate. GSH appears to protect against coumarin-induced toxicity possibly by the formation of conjugates with the toxic coumarin metabolite(s).     

Targeted and non‐targeted metabolite identification of MAM‐2201 in human,mouse, and rat hepatocytes

MAM‐2201 is a fluorinated naphthoylindole synthetic cannabinoid with potent psychoactive properties that has been detected as an active ingredient in herbal incense blends. To gain a greater understanding of MAM‐2201 metabolism and to compare its metabolic fate in humans with those in animals, the metabolism of MAM‐2201 in human, mouse, and rat hepatocytes was investigated using liquid chromatography–high‐resolution mass spectrometry combined with targeted and non‐targeted metabolite profiling approaches. Nineteen phase I metabolites (M1–M19) reported previously in human liver microsomes and 13 novel metabolites were identified in human, mouse, and rat hepatocytes: 1 phase I metabolite (M20) and 12 phase II metabolites including 6 glucuronides (G1–G6), 1 sulfate (S1), and 5 glutathione (GSH) conjugates (GS1–GS5) of MAM‐2201 metabolites. G3 was human‐specific, but M20, G1, G2, and 5 GSH conjugates were rat‐specific, indicating species‐related differences in MAM‐2201 metabolism. The findings in the present study can be useful for the experimental design and assessment of metabolism‐mediated toxic risk of MAM‐2201.     

Dioxin-Like and Non-Dioxin-Like Toxic Effects of Polychlorinated Biphenyls (PCBs): Implications For Risk Assessment

Polychlorinated biphenyls (PCBs) are persistent, bioaccumulative, and toxic contaminants in the environment. Individual PCB congeners exhibit different phy sicochemical properties and biological activities that result in different environmental distributions and toxicity profiles. The variable composition of PCB residues in environmental matrices and their different mechanisms of toxicity complicate the development of scientifically based regulations for the risk assessment. In this article various approaches for the assessment of risks of PCBs have been critically examined. Recent developments in the toxic equivalency factor (TEF) approach for the assessment of toxic effects due to dioxin-like PCBs have been examined. PCB exposure studies that describe non-dioxin-like toxic effects, particularly neurobehavioral effects and their effective doses in animals were compiled. A comparative assessment of effective doses for dioxin-like and non-dioxin-like effects by PCBs has been made to evaluate the relative significance of non-ortho-and ortho-substituted PCBs in risk assessment. Using mink as an example, relative merits and implications of using TEF and total PCB approaches for assessing the potential for toxic effects in wildlife was examined. There are several advantages and limitations associated with each method used for PCB risk assessment. Toxic effects due to coplanar PCBs occur at relatively smaller concentrations than those due to non-dioxin-like PCBs and therefore the TEF approach derives the risk assessment of PCBs, in the environment. The need for the refinement of TEF approach for more accurate assessment of risks is discussed.     

Cell membrane-related toxic responses and disruption of intercellular communication in PCB mechanisms of toxicity: A review

An understanding of polychlorinated biphenyl (PCB) congener-specific effects on cell membrane and intercellular communication is important within the studies of PCB absorption, organ-related PCB accumulation and exertion of toxic responses. Toxic potential of PCBs is linked to various deleterious effects on human health, including neurotoxicity, immunotoxicity, reproductive toxicity and genotoxicity and, recently in 2016 International Agency for Research on Cancer (IARC) has upgraded the classification of PCBs to Group 1 “Carcinogenic to humans.” Proposed mechanisms of aforementioned PCBs adverse effects at cellular membrane level are: (i) downregulation of gap junction intercellular communication and/or connexins; (ii) compromised membrane integrity; and (iii) altered tight junction barrier function. This study, based on an extensive literature survey, shows the progress in scientific research of each of these three levels with the aim of pointing out the earliest toxic events of PCBs, which can result in serious cell/tissue/organ damage.     

Sequential oxidation and glutathione addition to 1,4-benzoquinone: correlation of toxicity with increased glutathione substitution

The chemical reaction of 1,4-benzoquinone with glutathione results in the formation of adducts that exhibit increasing degrees of glutathione substitution. Purification of these adducts and analysis by 1H and 13C nuclear magnetic resonance spectroscopy revealed the products of the reaction to be 2-(glutathion-S-yl)hydroquinone; 2,3-(diglutathion-S-yl)hydroquinone; 2,5-(diglutathion-S-yl)hydroquinone; 2,6(diglutathion-S-yl)hydroquinone; 2,3,5-(triglutathion-S-yl)hydroquinone; and 2,3,5,6-(tetraglutatathion-S-yl)hydroquinone. The initial conjugation of 1,4-benzoquinone with glutathione did not significantly affect the oxidation potential of the compound. However, subsequent oxidation and glutathione addition resulted in the formation of conjugates that, dependent upon the position of addition, become increasingly more difficult to oxidize. Increased glutathione substitutions, which resulted in an increase in oxidation potentials, paradoxically resulted in enhanced nephrotoxicity. The triglutathion-S-yl conjugate was the most potent nephrotoxicant; the diglutathion-S-yl conjugates exhibited similar degrees of nephrotoxicity; the mono- and tetraglutathion-S-yl conjugates were not toxic. Thus, with the exception of the fully substituted isomer, the severity of renal necrosis correlated with the extent of glutathione substitution. The lack of toxicity of the fully substituted isomer is probably a consequence of its inability to alkylate tissue components. Thus, the conjugation of glutathione with quinones does not necessarily result in detoxification, even when the resulting conjugates are more stable to oxidation. The inhibition of gamma-glutamyl transpeptidase by AT-125 protected against 2,3,5-(triglutathion-S-yl)hydroquinone-mediated nephrotoxicity. It is suggested that other extra-renal sites expressing relatively high levels of gamma-glutamyl transpeptidase might therefore also be susceptible to hydroquinone-linked glutathione conjugate toxicity. This pathway might also contribute to the carcinogenicity and mutagenicity of certain quinones.     

Glutathione-dependent toxicity.

1. Recent studies show that glutathione conjugate formation is an important bioactivation mechanism for several groups of compounds with implications for organ-selective toxicity and carcinogenicity. 2. Vicinal dihaloalkanes, such as 1,2-dihaloethanes, yield S-(2-haloalkyl)glutathione conjugates that give rise to highly electrophilic episulphonium ions, which are involved in the cytotoxicity and mutagenicity of 1,2-dihaloethanes. 3. Nephrotoxic haloalkenes are metabolized to S-(haloalkenyl)- or S-(haloalkyl)-glutathione conjugates which, after metabolism to the corresponding cysteine conjugates, are bioactivated by renal cysteine conjugate beta-lyase to yield cytotoxic or mutagenic metabolites. 4. Finally, hepatic glutathione conjugate formation with hydroquinones and aminophenols yields conjugates that are directed to gamma-glutamyltransferase-rich tissues, such as the kidney, where they undergo alkylation or redox cycling reactions, or both, that cause organ-selective damage.     

"Ecstasy"-induced toxicity in SH-SY5Y differentiated cells: role of hyperthermia and metabolites

3,4-Methylenedioxymethamphetamine (MDMA; “ecstasy”) is a recreational hallucinogenic drug of abuse known to elicit neurotoxic properties. Hepatic formation of neurotoxic metabolites is thought to play a major role in MDMA-related neurotoxicity, though the mechanisms involved are still unclear. Here, we studied the neurotoxicity mechanisms and stability of MDMA and 6 of its major human metabolites, namely α-methyldopamine (α-MeDA) and N-methyl-α-methyldopamine (N-Me-α-MeDA) and their correspondent glutathione (GSH) and N-acetyl-cysteine (NAC) conjugates, under normothermic (37 °C) or hyperthermic conditions (40 °C), using cultured SH-SY5Y differentiated cells. We showed that MDMA metabolites exhibited toxicity to SH-SY5Y differentiated cells, being the GSH and NAC conjugates more toxic than their catecholic precursors and MDMA. Furthermore, whereas the toxicity of the catechol metabolites was potentiated by hyperthermia, NAC-conjugated metabolites revealed higher toxicity under normothermia and GSH-conjugated metabolites-induced toxicity was temperature-independent. Moreover, a time-dependent decrease in extracellular concentration of MDMA metabolites was observed, which was potentiated by hyperthermia. The antioxidant NAC significantly protected against the neurotoxic effects of MDMA metabolites. MDMA metabolites increased intracellular glutathione levels, though depletion in thiol content was observed in MDMA-exposed cells. Finally, the neurotoxic effects induced by the MDMA metabolite N-Me-α-MeDA involved caspase 3 activation. In conclusion, this study evaluated the stability of MDMA metabolites in vitro, and demonstrated that the catechol MDMA metabolites and their GSH and NAC conjugates, rather than MDMA itself, exhibited neurotoxic actions in SH-SY5Y differentiated cells, which were differently affected by hyperthermia, thus highlighting a major role for reactive metabolites and hyperthermia in MDMA’s neurotoxicity.     

Assessment of the metabolism and intrinsic reactivity of a novel catechol metabolite

PH-302 ( 1) demonstrates potent inhibitory activity against the inducible form of nitric oxide synthase (iNOS). The primary metabolite of PH-302 is a catechol ( 2) resulting from oxidative demethylenation of the methylenedioxyphenyl moiety by cytochrome P450 3A4. Concerns regarding subsequent two-electron oxidation of 2 to an electrophilic quinone species and the potential for resulting toxicity prompted additional studies to examine the reactivity and metabolic fate of this metabolite. Contrary to literature reports of catechol reactivity, 2 appeared to be resistant to quinone formation in human liver microsomal incubations, as determined by the lack of detectable glutathione (GSH) adducts and no covalent binding to microsomal proteins. In addition, 2 showed no evidence of depletion of intracellular glutathione or cytotoxicity at concentrations up to 1 mM in primary human and rat hepatocytes. In the presence of tyrosinase, spectral evidence indicated that 2 was oxidized to the ortho-quinone, and upon incubation in the presence of GSH, two conjugates were detected and characterized by LC/MS/MS. Lastly, the metabolic pathways of 2 were investigated in rat and human hepatocytes and found to be similar, proceeding via glucuronidation, sulfation, and methylation of the catechol. Collectively, these studies demonstrate that 2 appears to be resistant to further oxidation to quinone in liver microsomes, as well as spontaneous redox cycling, while the formation of phase II metabolites in hepatocytes suggests that multiple detoxication pathways may provide added protection against toxicity in the liver.     

Prediction of the health effects of polychlorinated biphenyls (PCBs) and their metabolites using quantitative structure-activity relationship (QSAR)

Polychlorinated biphenyls (PCBs) are a group of 209 persistent environmental contaminants that are slightly different but structurally related. PCBs are known to induce a variety of health effects and often have been toxicologically tested as complex commercial mixtures (Aroclors) but environmental exposure occurs separately to a small number of specific congeners. Recently, the Third National Report on Human Exposures to Environmental Chemicals, an assessment of exposure data of the National Health and Nutrition Examination Survey (NHANES), identified 35 individual PCB congeners in the U.S. population. These types of findings necessitate the toxicity evaluation of individual congeners but adequate toxicity data for most individual PCB congeners are not available. Due to this, a quantitative structure-activity relationship (QSAR) approach was used to assess the potential mutagenesis and carcinogenesis of individual congeners and their possible metabolites. The predictions were analyzed to define the underlying generalizations between the parent PCBs, their metabolites, and some important toxicological endpoints. This analysis reveals that (1) mono and di-chlorinated PCBs and their metabolites can be potential mutagens; (2) PCB benzoquinone metabolites could be carcinogenic but the weight of evidence is poor. These results support the hypothesis that environmental exposure to some PCBs and/or their metabolites could produce mutagenicity and/or carcinogenicity. Hence, these data should be considered as priority toxicological testing data needs. As with all computational toxicology analytical findings, these conclusions must yield to empirical data as they become available.     

Identification of quinone imine containing glutathione conjugates of diclofenac in rat bile

High-resolution accurate MS with an LTQ-Orbitrap was used to identify quinone imine metabolites derived from the 5-hydroxy (5-OH) and 4 prime-hydroxy (4'-OH) glutathione conjugates of diclofenac in rat bile. The initial quinone imine metabolites formed by oxidation of diclofenac have been postulated to be reactive intermediates potentially involved in diclofenac-mediated hepatotoxicity; while these metabolites could be formed using in vitro systems, they have never been detected in vivo. This report describes the identification of secondary quinone imine metabolites derived from 5-OH and 4'-OH diclofenac glutathione conjugates in rat bile. To verify the proposed structures, the diclofenac quinone imine GSH conjugate standards were prepared synthetically and enzymatically. The novel metabolite peaks displayed the identical retention times, accurate mass MS/MS spectra, and the fragmentation patterns as the corresponding authentic standards. The formation of these secondary quinone metabolites occurs only under conditions where bile salt homeostasis was experimentally altered. Standard practice in biliary excretion experiments using bile duct-cannulated rats includes infusion of taurocholic acid and/or other bile acids to replace those lost due to continuous collection of bile; for this experiment, the rats received no replacement bile acid infusion. High-resolution accurate mass spectrometry data and comparison with chemically and enzymatically prepared quinone imines of diclofenac glutathione conjugates support the identification of these metabolites. A mechanism for the formation of these reactive quinone imine containing glutathione conjugates of diclofenac is proposed.     

Morphological alterations of Vero cell exposed to coplanar PCB 126 and noncoplanar PCB 153

Polychlorinated biphenyls (PCBs) are widespread, persistent environmental contaminants that display a complex spectrum of toxicological properties. Exposure to PCBs has been associated with morphological anomalies in cell cultures. However, most mechanistic studies of PCBs' toxic activity have been focused on coplanar congeners. It is of importance to determine whether PCB treatment would influence cell configuration and whether these changes would depend on the structural characteristics of PCBs. In this study, we investigated cell morphological alteration in Vero cell cultures after exposure to coplanar PCB 126 and noncoplanar PCB 153. The survival of Vero cells was measured through the MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) test. Cytotoxicity results suggested that PCB congeners had a toxic, antiproliferative effect on Vero cells. Morphological studies described structural modifications and provided evidence that apoptosis might be the main cell death pathway in PCB 153‐treated cells. The comparison between PCB 126 and PCB 153 indicated that the cell death mechanisms involved in coplanar or noncoplanar PCB congener exposure were different in Vero cells. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.