The effect of black cohosh extract and risedronate coadministration on bone health in an ovariectomized rat model.

Preparations of black cohosh extract are sold as dietary supplements marketed to relieve the vasomotor symptoms of menopause, and some studies suggest it may protect against postmenopausal bone loss. Postmenopausal women are also frequently prescribed bisphosphonates, such as risedronate, to prevent osteoporotic bone loss. However, the pharmacodynamic interactions between these compounds when taken together is not known. To investigate possible interactions, 6-month-old, female Sprague-Dawley rats underwent bilateral ovariectomy or sham surgery and were treated for 24 weeks with either vehicle, ethinyl estradiol, risedronate, black cohosh extract or coadministration of risedronate and black cohosh extract, at low or high doses. Bone mineral density (BMD) of the femur, tibia, and lumbar vertebrae was then measured by dual-energy X-ray absorptiometry (DEXA) at weeks 0, 8, 16, and 24. A high dose of risedronate significantly increased BMD of the femur and vertebrae, while black cohosh extract had no significant effect on BMD individually and minimal effects upon coadministration with risedronate. Under these experimental conditions, black cohosh extract alone had no effect on BMD, nor did it negatively impact the BMD-enhancing properties of risedronate.

Effect of urinary pH upon the renal toxicity of melamine and cyanuric acid.

In 2007, dietary exposure to "scrap melamine' resulted in the death of a large number of cats and dogs, which was attributed to the formation of melamine cyanurate crystals in their kidneys. In this study, we investigated if changes in urinary pH could diminish the renal toxicity associated with exposure to combinations of melamine and cyanuric acid. Female Sprague-Dawley rats were treated for three days with suspensions of melamine and cyanuric acid at doses that were expected to induce renal toxicity. Dosing was then discontinued and the rats were treated for seven days with drinking water solutions (i.e., ammonium chloride and sodium bicarbonate) that would alter urinary pH. The urinary pH of rats administered ammonium chloride drinking water decreased from pH 6.0-6.2 to pH 5.1-5.2. This was accompanied by a decrease in the incidence of melamine cyanurate crystals in the kidneys and a decrease in the incidence of renal lesions. These data suggest that acidification of urine may help overcome the renal toxicities associated with the formation of melamine cyanurate crystals in the kidney.

Mutagenicity of acrylamide and glycidamide in human TP53 knock-in (Hupki) mouse embryo fibroblasts.

Acrylamide is a suspected human carcinogen formed during high-temperature cooking of starch-rich foods. It is metabolised by cytochrome P450 2E1 to its reactive metabolite glycidamide, which forms pre-mutagenic DNA adducts. Using the human TP53 knock-in (Hupki) mouse embryo fibroblasts (HUFs) immortalisation assay (HIMA), acrylamide- and glycidamide-induced mutagenesis was studied in the tumour suppressor gene TP53. Selected immortalised HUF clones were also subjected to next-generation sequencing to determine mutations across the whole genome. The TP53-mutant frequency after glycidamide exposure (1.1 mM for 24 h, n = 198) was 9% compared with 0% in cultures treated with acrylamide [1.5 (n = 24) or 3 mM (n = 6) for 48 h] and untreated vehicle (water) controls (n = 36). Most glycidamide-induced mutations occurred at adenines with A > T/T > A and A > G/T > C mutations being the most common types. Mutations induced by glycidamide occurred at specific TP53 codons that have also been found to be mutated in human tumours (i.e., breast, ovary, colorectal, and lung) previously associated with acrylamide exposure. The spectrum of TP53 mutations was further reflected by the mutations detected by whole-genome sequencing (WGS) and a distinct WGS mutational signature was found in HUF clones treated with glycidamide that was again characterised by A > G/T > C and A > T/T > A mutations. The WGS mutational signature showed similarities with COSMIC mutational signatures SBS3 and 25 previously found in human tumours (e.g., breast and ovary), while the adenine component was similar to COSMIC SBS4 found mostly in smokers' lung cancer. In contrast, in acrylamide-treated HUF clones, only culture-related background WGS mutational signatures were observed. In summary, the results of the present study suggest that glycidamide may be involved in the development of breast, ovarian, and lung cancer.

Use of a physiologically-based pharmacokinetic model to explore the potential disparity in nicotine disposition between adult and adolescent nonhuman primates.

The widespread use and high abuse liability of tobacco products has received considerable public health attention, in particular for youth, who are vulnerable to nicotine addiction. In this study, adult and adolescent squirrel monkeys were used to evaluate age-related metabolism and pharmacokinetics of nicotine after intravenous administration. A physiologically-based pharmacokinetic (PBPK) model was created to characterize the pharmacokinetic behaviors of nicotine and its metabolites, cotinine, trans-3'-hydroxycotinine (3'-OH cotinine), and trans-3'-hydroxycotinine glucuronide (3'-OH cotinine glucuronide) for both adult and adolescent squirrel monkeys. The PBPK nicotine model was first calibrated for adult squirrel monkeys utilizing in vitro nicotine metabolic data, plasma concentration-time profiles and cumulative urinary excretion data for nicotine and metabolites. Further model refinement was conducted when the calibrated adult model was scaled to the adolescents, because adolescents appeared to clear nicotine and cotinine more rapidly relative to adults. More specifically, the resultant model parameters representing systemic clearance of nicotine and cotinine for adolescent monkeys were approximately two- to three-fold of the adult values on a per body weight basis. The nonhuman primate PBPK model in general captured experimental observations that were used for both model calibration and evaluation, with acceptable performance metrics for precision and bias. The model also identified differences in nicotine pharmacokinetics between adolescent and adult nonhuman primates which might also be present in humans.

1-Formyl-7-hydroxy-6,7-dihydro-5 H-pyrrolizine (1-CHO-DHP): A Potential Proximate Carcinogenic Metabolite of Pyrrolizidine Alkaloids.

Pyrrolizidine alkaloids (PAs) are phytochemicals present in more than 6000 plant species worldwide; about half of the PAs are hepatotoxic, genotoxic, and carcinogenic. Because of their wide exposure and carcinogenicity, the International Programme on Chemical Safety (IPCS) concluded that PAs are a threat to human health and safety. We recently determined that PA-induced liver tumor initiation is mediated by a set of four (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5 H-pyrrolizine (DHP)-DNA adducts and proposed that these DHP-DNA adducts are biomarkers of PA exposure and liver tumor initiation. To validate the generality of this metabolic activation pathway and DHP-DNA adducts as biomarkers, it is significant to identify reactive metabolites associated with this metabolic activation pathway. Segall et al. ( Segall et al. ( 1984 ) Drug Metab. Dispos. 12 , 68 - 71 ) previously reported that 1-formyl-7-hydroxy-6,7-dihydro-5 H-pyrrolizine (1-CHO-DHP) is generated from the metabolism of senecionine by mouse liver microsomes. In the present study, we examined the metabolism of seven hepatocarcinogenic PAs (senecionine, intermedine, retrorsine, riddelliine, DHR, heliotrine, and senkirkine) and one noncarcinogenic PA (platyphylline) by human, rat, and mouse liver microsomes. 1-CHO-DHP was identified as a common metabolite from the metabolism of these hepatotoxic PAs, but not from platyphylline. Incubation of 1-CHO-DHP with HepG2 and A549 cells produced the same set of DHP-DNA adducts, which were identified by both LC/MS MRM mode and selected ion monitoring analyses through comparison to synthetic standards. In the incubation medium of 1-CHO-DHP treated HepG2 cells, both DHP and 7-cysteine-DHP were formed, which were capable of binding to cellular DNA to produce DHP-DNA adducts. These results suggest that 1-CHO-DHP is a proximate DNA metabolite of genotoxic and carcinogenic PAs.

Effects of human sulfotransferases on the cytotoxicity of 12-hydroxynevirapine.

Nevirapine, a non-nucleoside reverse transcriptase inhibitor used for the treatment of AIDS, can cause serious skin rashes and hepatotoxicity. Previous studies have indicated that the benzylic sulfate 12-sulfoxynevirapine, the formation of which is catalyzed by human sulfotransferases (SULTs), may play a causative role in these toxicities. To characterize better the role of 12-sulfoxynevirapine in nevirapine-induced cytotoxicity, the ability of 12 expressed human SULT isoforms to conjugate 12-hydroxynevirapine was assessed. Of the 12 human SULTs, no detectable 12-sulfoxynevirapine was observed with SULT1A3, SULT1C2, SULT1C3, SULT2B1, SULT4A1, or SULT6B1. As determined by the Vmax/Km ratio, SULT2A1 had the highest overall 12-hydoxynevirapine sulfonation activity; lower activities were observed with SULT1A1, SULT1A2, SULT1B1, SULT1C4, and SULT1E1. Incubation of 12-sulfoxynevirapine with glutathione and cysteine led to adduct formation; lower yields were obtained with deoxynucleosides. 12-Hydroxynevirapine was more cytotoxic than nevirapine to TK6, TK6/SULT vector, and TK6/SULT2A1 cells. With nevirapine, there was no difference in cytotoxicity among the three cell lines, whereas with 12-hydroxynevirapine, TK6/SULT2A1 cells were more resistant than TK6 and TK6/SULT vector cells. Co-incubation of 12-hydroxynevirapine with the competitive SULT2A1 substrate dehydroepiandrosterone decreased the level of 12-sulfoxynevirapine and increased the cytotoxicity in TK6/SULT2A1 cells. These data demonstrate that although 12-sulfoxynevirapine reacts with nucleophiles to form adducts, sulfonation of 12-hydroxynevirapine decreases the cytotoxicity of 12-hydroxynevirapine in TK6 cells.

A simple and highly sensitive UPLC-ESI-MS/MS method for the simultaneous quantification of nicotine, cotinine, and the tobacco-specific carcinogens N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in serum samples.

According to the World Health Organization, the consumption of tobacco products is the single largest cause of preventable deaths in the world, exceeding the total aggregated number of deaths caused by diseases such as AIDS, tuberculosis, and malaria. An important element in the evaluation of the health risks associated with the consumption of tobacco products is the assessment of the internal exposure to the tobacco constituents responsible for their addictive (e.g. nicotine) and carcinogenic (e.g. N-nitrosamines such as NNN and NNK) properties. However, the assessment of the serum levels of these compounds is often challenging from an analytical standpoint, in particular when limited sample volumes are available and low detection limits are required. Currently available analytical methods often rely on complex multi-step sample preparation procedures, which are prone to low analyte recoveries and ex-vivo contamination due to the ubiquitous nature of these compounds as background contaminants. In order to circumvent these problems, we report a facile and highly sensitive method for the simultaneous quantification of nicotine, cotinine, NNN, and NNK in serum samples. The method relies on a simple "one pot" liquid-liquid extraction procedure and isotope dilution ultra-high pressure (UPLC) hydrophilic interaction liquid chromatography (HILIC) coupled with tandem mass spectrometry. The method requires only 10µL of serum and presents a limit of quantification of 0.02nmol (3000pg/mL) nicotine, 0.6pmol (100pg/mL) cotinine, 0.05pmol NNK (10pg/mL), and 0.06pmol NNN (10pg/mL), making it appropriate for pharmacokinetic evaluations.

Cytochrome P450-mediated metabolism of triclosan attenuates its cytotoxicity in hepatic cells.

Triclosan is a widely used broad-spectrum anti-bacterial agent. The objectives of this study were to identify which cytochrome P450 (CYP) isoforms metabolize triclosan and to examine the effects of CYP-mediated metabolism on triclosan-induced cytotoxicity. A panel of HepG2-derived cell lines was established, each of which overexpressed a single CYP isoform, including CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP3A7, CYP4A11, and CYP4B1. The extent of triclosan metabolism by each CYP was assessed by reversed-phase high-performance liquid chromatography with online radiochemical detection. Seven isoforms were capable of metabolizing triclosan, with the order of activity being CYP1A2 > CYP2B6 > CYP2C19 > CYP2D6 ≈ CYP1B1 > CYP2C18 ≈ CYP1A1. The remaining 11 isoforms (CYP2A6, CYP2A7, CYP2A13, CYP2C8, CYP2C9, CYP2E1, CYP3A4, CYP3A5, CYP3A7, CYP4A11, and CYP4B1) had little or no activity toward triclosan. Three metabolites were detected: 2,4-dichlorophenol, 4-chlorocatechol, and 5'-hydroxytriclosan. Consistent with the in vitro screening data, triclosan was extensively metabolized in HepG2 cells overexpressing CYP1A2, CYP2B6, CYP2C19, CYP2D6, and CYP2C18, and these cells were much more resistant to triclosan-induced cytotoxicity compared to vector cells, suggesting that CYP-mediated metabolism of triclosan attenuated its cytotoxicity. In addition, 2,4-dichlorophenol and 4-chlorocatechol were less toxic than triclosan to HepG2/vector cells. Conjugation of triclosan, catalyzed by human glucuronosyltransferases (UGTs) and sulfotransferases (SULTs), also occurred in HepG2/CYP-overexpressing cells and primary human hepatocytes, with a greater extent of conjugation being associated with higher cell viability. Co-administration of triclosan with UGT or SULT inhibitors led to greater cytotoxicity in HepG2 cells and primary human hepatocytes, indicating that glucuronidation and sulfonation of triclosan are detoxification pathways. Among the 18 CYP-overexpressing cell lines, an inverse correlation was observed between cell viability and the level of triclosan in the culture medium. In conclusion, human CYP isoforms that metabolize triclosan were identified, and the metabolism of triclosan by CYPs, UGTs, and SULTs decreased its cytotoxicity in hepatic cells.

Simple and rapid quantification of brominated vegetable oil in commercial soft drinks by LC-MS.

We report here a simple and rapid method for the quantification of brominated vegetable oil (BVO) in soft drinks based upon liquid chromatography-electrospray ionization mass spectrometry. Unlike previously reported methods, this novel method does not require hydrolysis, extraction or derivatization steps, but rather a simple "dilute and shoot" sample preparation. The quantification is conducted by mass spectrometry in selected ion recording mode and a single point standard addition procedure. The method was validated in the range of 5-25µg/mL BVO, encompassing the legal limit of 15µg/mL established by the US FDA for fruit-flavored beverages in the US market. The method was characterized by excellent intra- and inter-assay accuracy (97.3-103.4%) and very low imprecision [0.5-3.6% (RSD)]. The direct nature of the quantification, simplicity, and excellent statistical performance of this methodology constitute clear advantages in relation to previously published methods for the analysis of BVO in soft drinks.

The role of fibrinogen glycation in ATTR: evidence for chaperone activity loss in disease.

Transthyretin amyloidosis (ATTR) belongs to a class of disorders caused by protein misfolding and aggregation. ATTR is a disabling disorder of autosomal dominant trait, where transthyretin (TTR) forms amyloid deposits in different organs, causing dysfunction of the peripheral nervous system. We previously discovered that amyloid fibrils from ATTR patients are glycated by methylglyoxal. Even though no consensus has been reached about the actual role of methylglyoxal-derived advanced glycation end-products in amyloid diseases, evidence collected so far points to a role for protein glycation in conformational abnormalities, being ubiquitously found in amyloid deposits in Alzheimer's disease, dialysis-related amyloidosis and Parkinson's diseases. Human fibrinogen, an extracellular chaperone, was reported to specifically interact with a wide spectrum of stressed proteins and suppress their aggregation, being an interacting protein with TTR. Fibrinogen is differentially glycated in ATTR, leading to its chaperone activity loss. Here we show the existence of a proteostasis imbalance in ATTR linked to fibrinogen glycation by methylglyoxal.

Hydrogen peroxide regulates cell adhesion through the redox sensor RPSA.

To become metastatic, a tumor cell must acquire new adhesion properties that allow migration into the surrounding connective tissue, transmigration across endothelial cells to reach the blood stream and, at the site of metastasis, adhesion to endothelial cells and transmigration to colonize a new tissue. Hydrogen peroxide (H2O2) is a redox signaling molecule produced in tumor cell microenvironment with high relevance for tumor development. However, the molecular mechanisms regulated by H2O2 in tumor cells are still poorly known. The identification of H2O2-target proteins in tumor cells and the understanding of their role in tumor cell adhesion are essential for the development of novel redox-based therapies for cancer. In this paper, we identified Ribosomal Protein SA (RPSA) as a target of H2O2 and showed that RPSA in the oxidized state accumulates in clusters that contain specific adhesion molecules. Furthermore, we showed that RPSA oxidation improves cell adhesion efficiency to laminin in vitro and promotes cell extravasation in vivo. Our results unravel a new mechanism for H2O2-dependent modulation of cell adhesion properties and identify RPSA as the H2O2 sensor in this process. This work indicates that high levels of RPSA expression might confer a selective advantage to tumor cells in an oxidative environment.

Absorption and metabolism of triclosan after application to the skin of B6C3F1 mice.

Triclosan is used as an antimicrobial agent in personal care products, household items, medical devices, and clinical settings. Humans can receive lifelong exposures to triclosan; however, data on the toxicity and carcinogenicity after topical application are lacking. This study determined the absorption, distribution, metabolism, and excretion of triclosan after application to the skin of B6C3F1 mice. [(14)C(U)]triclosan (10 or 100 mg triclosan/kg body weight) was administered topically to mice in two separate experiments: a vehicle selection experiment using propylene glycol, ethanol, and a generic cosmetic cream, and a toxicokinetic experiment. Mice were killed up to 72 h after triclosan administration, and excreta and tissues were analyzed for radioactivity. Ethanol had the best properties of the vehicles evaluated. Maximum absorption was obtained at approximately 12 h after dosing. Radioactivity appeared in the excreta and in all tissues examined, with the highest levels in the gall bladder and the lowest levels in the brain. Triclosan was metabolized to triclosan sulfate, triclosan glucuronide, 2,4-dichlorophenol, and hydroxytriclosan. The metabolite profile was tissue-dependent and the predominant route of excretion was fecal. The AUC(0-∞) and the Cmax of plasma and liver in females were greater than those in males. Slightly lower absorption was observed in mice with Elizabethan collars.

Metabolic activation of 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine and DNA adduct formation depends on p53: Studies in Trp53(+/+),Trp53(+/-) and Trp53(-/-) mice.

The expression of the tumor suppressor p53 can influence the bioactivation of, and DNA damage induced by, the environmental carcinogen benzo[a]pyrene, indicating a role for p53 in its cytochrome P450 (CYP)-mediated biotransformation. The carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which is formed during the cooking of food, is also metabolically activated by CYP enzymes, particularly CYP1A2. We investigated the potential role of p53 in PhIP metabolism in vivo by treating Trp53(+/+), Trp53(+/-) and Trp53(-/-) mice with a single oral dose of 50 mg/kg body weight PhIP. N-(Deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP-C8-dG) levels in DNA, measured by liquid chromatography-tandem mass spectrometry, were significantly lower in liver, colon, forestomach and glandular stomach of Trp53(-/-) mice compared to Trp53(+/+) mice. Lower PhIP-DNA adduct levels in the livers of Trp53(-/-) mice correlated with lower Cyp1a2 enzyme activity (measured by methoxyresorufin-O-demethylase activity) in these animals. Interestingly, PhIP-DNA adduct levels were significantly higher in kidney and bladder of Trp53(-/-) mice compared to Trp53(+/+) mice, which was accompanied by higher sulfotransferase (Sult) 1a1 protein levels and increased Sult1a1 enzyme activity (measured by 2-naphthylsulfate formation from 2-naphthol) in kidneys of these animals. Our study demonstrates a role for p53 in the metabolism of PhIP in vivo, extending previous results on a novel role for p53 in xenobiotic metabolism. Our results also indicate that the impact of p53 on PhIP biotransformation is tissue-dependent and that in addition to Cyp1a enzymes, Sult1a1 can contribute to PhIP-DNA adduct formation.

Transthyretin Amyloidosis: Chaperone Concentration Changes and Increased Proteolysis in the Pathway to Disease.

Transthyretin amyloidosis is a conformational pathology characterized by the extracellular formation of amyloid deposits and the progressive impairment of the peripheral nervous system. Point mutations in this tetrameric plasma protein decrease its stability and are linked to disease onset and progression. Since non-mutated transthyretin also forms amyloid in systemic senile amyloidosis and some mutation bearers are asymptomatic throughout their lives, non-genetic factors must also be involved in transthyretin amyloidosis. We discovered, using a differential proteomics approach, that extracellular chaperones such as fibrinogen, clusterin, haptoglobin, alpha-1-anti-trypsin and 2-macroglobulin are overrepresented in transthyretin amyloidosis. Our data shows that a complex network of extracellular chaperones are over represented in human plasma and we speculate that they act synergistically to cope with amyloid prone proteins. Proteostasis may thus be as important as point mutations in transthyretin amyloidosis.

Effects of oral exposure to bisphenol A on gene expression and global genomic DNA methylation in the prostate, female mammary gland, and uterus of NCTR Sprague-Dawley rats.

Bisphenol A (BPA), an industrial chemical used in the manufacture of polycarbonate and epoxy resins, binds to the nuclear estrogen receptor with an affinity 4-5 orders of magnitude lower than that of estradiol. We reported previously that "high BPA" [100,000 and 300,000 µg/kg body weight (bw)/day], but not "low BPA" (2.5-2700 µg/kg bw/day), induced clear adverse effects in NCTR Sprague-Dawley rats gavaged daily from gestation day 6 through postnatal day (PND) 90. The "high BPA" effects partially overlapped those of ethinyl estradiol (EE2, 0.5 and 5.0 µg/kg bw/day). To evaluate further the potential of "low BPA" to induce biological effects, here we assessed the global genomic DNA methylation and gene expression in the prostate and female mammary glands, tissues identified previously as potential targets of BPA, and uterus, a sensitive estrogen-responsive tissue. Both doses of EE2 modulated gene expression, including of known estrogen-responsive genes, and PND 4 global gene expression data showed a partial overlap of the "high BPA" effects with those of EE2. The "low BPA" doses modulated the expression of several genes; however, the absence of a dose response reduces the likelihood that these changes were causally linked to the treatment. These results are consistent with the toxicity outcomes.

Reaction of dehydropyrrolizidine alkaloids with valine and hemoglobin.

Pyrrolizidine alkaloid-containing plants are probably the most common poisonous plants affecting livestock, wildlife, and humans. Pyrrolizidine alkaloids exert toxicity through metabolism to dehydropyrrolizidine alkaloids that bind to cellular protein and DNA, leading to hepatotoxicity, genotoxicity, and tumorigenicity. To date, it is not clear how dehydropyrrolizidine alkaloids bind to cellular constituents, including amino acids and proteins, resulting in toxicity. Metabolism of carcinogenic monocrotaline, riddelliine, and heliotrine produces dehydromonocrotaline, dehyroriddelliine, and dehydroheliotrine, respectively, as primary reactive metabolites. In this study, we report that reaction of dehydromonocrotaline with valine generated four highly unstable 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived valine (DHP-valine) adducts. For structural elucidation, DHP-valine adducts were derivatized with phenyl isothiocyanate (PITC) to DHP-valine-PITC products. After HPLC separation, their structures were characterized by mass spectrometry, UV-visible spectrophotometry, (1)H NMR, and (1)H-(1)H COSY NMR spectral analysis. Two DHP-valine-PITC adducts, designated as DHP-valine-PITC-1 and DHP-valine-PITC-3, had the amino group of valine linked to the C7 position of the necine base, and the other two DHP-valine-PITC products, DHP-valine-PITC-2 and DHP-valine-PITC-4, linked to the C9 position of the necine base. DHP-valine-PITC-1 was interconvertible with DHP-valine-PITC-3, and DHP-valine-PITC-2 was interconvertible with DHP-valine-PITC-4. Reaction of dehydroriddelliine and dehydroheliotrine with valine provided similar results. However, reaction of valine and dehydroretronecine (DHR) under similar experimental conditions did not produce DHP-valine adducts. Reaction of dehydromonocrotaline with rat hemoglobin followed by derivatization with PITC also generated the same four DHP-valine-PITC adducts. This represents the first full structural elucidation of protein conjugated pyrrolic adducts formed from reaction of dehydropyrrolizidine alkaloids with an amino acid (valine). In addition, it was found that DHP-valine-2 and DHP-valine-4, with the valine amino group linked at the C7 position of the necine base, can lose the valine moiety to form DHP.

Exceptionally long-term persistence of DNA adducts formed by carcinogenic aristolochic acid I in renal tissue from patients with aristolochic acid nephropathy.

Aristolochic acid (AA) causes aristolochic acid nephropathy (AAN), first described in women in Belgium accidently prescribed Aristolochia fangchi in a slimming treatment, and also Balkan endemic nephropathy (BEN), through probable dietary contamination with Aristolochia clematitis seeds. Both nephropathies have a high risk of urothelial cancer, with AA being the causative agent. In tissues of AAN and BEN patients, a distinct DNA adduct, 7-(deoxyadenosin-N6-yl)-aristolactam I (dA-AAI), has been detected. DNA adducts can be removed through DNA repair, they can result in mutations through erroneous DNA replication or they can cause cell death. The dA-AAI adduct induces AT to TA transversions in the tumor-suppressor TP53 gene in experimental systems, matching TP53 mutations observed in urothelial tumors from AAN cancer cases. Using thin-layer chromatography 32P-postlabeling and mass spectrometric analysis we report the detection of dA-AAI in renal DNA from 11 Belgian AAN patients over 20 years after exposure to AA had ceased. Our results showed that dA-AAI is an established biomarker of AA exposure, and that this biomarker can be demonstrated to be persistent decades after a distinct AA exposure. Further, the persistence of dA-AAI adducts appears to be a critical determinant for the AA mutational fingerprint frequently found in oncogenes and tumor suppressor genes recently identified by whole genome sequencing of AA-associated urothelial tumors. The potential for exposure to AA worldwide is high; the unprecedented long-term persistence of dA-AAI provides a useful long-term biomarker of exposure and attests to the role of AA in human urothelial malignancy.

Toxicity evaluation of bisphenol A administered by gavage to Sprague Dawley rats from gestation day 6 through postnatal day 90.

Bisphenol A (BPA) is a high production volume industrial chemical to which there is widespread human oral exposure. Guideline studies used to set regulatory limits detected adverse effects only at doses well above human exposures and established a no-observed-adverse-effect level (NOAEL) of 5 mg/kg body weight (bw)/day. However, many reported animal studies link BPA to potentially adverse effects on multiple organ systems at doses below the NOAEL. The primary goals of the subchronic study reported here were to identify adverse effects induced by orally (gavage) administered BPA below the NOAEL, to characterize the dose response for such effects and to determine doses for a subsequent chronic study. Sprague Dawley rat dams were dosed daily from gestation day 6 until the start of labor, and their pups were directly dosed from day 1 after birth to termination. The primary focus was on seven equally spaced BPA doses (2.5-2700 µg/kg bw/day). Also included were a naïve control, two doses of ethinyl estradiol (EE2) to demonstrate the estrogen responsiveness of the animal model, and two high BPA doses (100,000 and 300,000 µg/kg bw/day) expected from guideline studies to produce adverse effects. Clear adverse effects of BPA, including depressed gestational and postnatal body weight gain, effects on the ovary (increased cystic follicles, depleted corpora lutea, and antral follicles), and serum hormones (increased serum estradiol and prolactin and decreased progesterone), were observed only at the two high doses of BPA. BPA-induced effects partially overlapped those induced by EE2, consistent with the known weak estrogenic activity of BPA.