Wolfgang Dekant

Comparative analysis of novel noninvasive renal biomarkers and metabonomic changes in a rat model of gentamicin nephrotoxicity.

Although early detection of toxicant induced kidney injury during drug development and chemical safety testing is still limited by the lack of sensitive and reliable biomarkers of nephrotoxicity, omics technologies have brought enormous opportunities for improved detection of toxicity and biomarker discovery. Thus, transcription profiling has led to the identification of several candidate kidney biomarkers such as kidney injury molecule (Kim-1), clusterin, lipocalin-2, and tissue inhibitor of metalloproteinase 1 (Timp-1), and metabonomic analysis of urine is increasingly used to indicate biochemical perturbations due to renal toxicity. This study was designed to assess the value of a combined (1)H-NMR and gas chromatography-mass spectrometry (GC-MS) metabonomics approach and a set of novel urinary protein markers for early detection of nephrotoxicity following treatment of male Wistar rats with gentamicin (60 and 120 mg/kg bw, s.c.) for 7 days. Time- and dose-dependent separation of gentamicin-treated animals from controls was observed by principal component analysis of (1)H-NMR and GC-MS data. The major metabolic alterations responsible for group separation were linked to the gut microflora, thus related to the pharmacology of the drug, and increased glucose in urine of gentamicin-treated animals, consistent with damage to the S(1) and S(2) proximal tubules, the primary sites for glucose reabsorption. Altered excretion of urinary protein biomarkers Kim-1 and lipocalin-2, but not Timp-1 and clusterin, was detected before marked changes in clinical chemistry parameters were evident. The early increase in urine, which correlated with enhanced gene and protein expression at the site of injury, provides further support for lipocalin-2 and Kim-1 as sensitive, noninvasive biomarkers of nephrotoxicity.

Endocrine effects of tetrabromobisphenol-A (TBBPA) in Wistar rats as tested in a one-generation reproduction study and a subacute toxicity study.

Endocrine effects of the brominated flame retardant tetrabromobisphenol-A (TBBPA) were studied in a one-generation reproduction assay in Wistar rats via repeated dietary exposure, applying eight dose groups at 0-3-10-30-100-300-1,000-3,000 mg/kg body weight/day (mkd). This design enables dose-response analysis and calculation of benchmark doses (BMDL). This reproduction study was preceded by a 28-day repeat dose subacute toxicity study, at 0-30-100-300 mkd. Major effects in the reproduction study included decreased circulating thyroxine (T4) with BMDLs of 31 (m) and 16 (f) mkd, and increased weight of testis and male pituitary (BMDLs of 0.5 and 0.6 mkd). The hypothyroxinemia correlated to a cluster of developmental parameters including delayed sexual development in females, decreased pup mortality, and effects on brainstem auditory evoked potentials [Lilienthal, H., Verwer, C.M., Van der Ven, L.T.M., Piersma, A.H., Vos, J.G., 2008. Neurobehavioral effects of tetrabromobisphenol A (TBBPA) in rats after pre- and postnatal exposure. Toxicology]. A second cluster of parameters in F1 animals was correlated to increased testis weight, and included female gonad weight, endometrium height, CYP19/aromatase activity in the ovary, and plasma testosterone levels in males. These two correlation clusters suggest a dual action of TBBPA. The only effects in the subacute study were decreased circulating T4 and increased T3 levels in males (BMDLs 48 and 124mkd), and non-significant trends for these parameters in females, suggesting that the other effects in the reproduction study were induced during development. Combined with data of human exposure to environmental TBBPA, the margin of exposure for highly exposed populations can be calculated at 2.6, and current use of TBBPA may therefore be a matter of concern for human health.

Performance of Novel Kidney Biomarkers in Preclinical Toxicity Studies

The kidney is one of the main targets of drug toxicity, but early detection of renal damage is often difficult. As part of the InnoMed PredTox project, a collaborative effort aimed at assessing the value of combining omics technologies with conventional toxicology methods for improved preclinical safety assessment, we evaluated the performance of a panel of novel kidney biomarkers in preclinical toxicity studies. Rats were treated with a reference nephrotoxin or one of several proprietary compounds that were dropped from drug development in part due to renal toxicity. Animals were dosed at two dose levels for 1, 3, and 14 days. Putative kidney markers, including kidney injury molecule-1 (Kim-1), lipocalin-2 (Lcn2), clusterin, and tissue inhibitor of metalloproteinases-1, were analyzed in kidney and urine using quantitative real-time PCR, ELISA, and immunohistochemistry. Changes in gene/protein expression generally correlated well with renal histopathological alterations and were frequently detected at earlier time points or at lower doses than the traditional clinical parameters blood urea nitrogen and serum creatinine. Urinary Kim-1 and clusterin reflected changes in gene/protein expression and histopathological alterations in the target organ in the absence of functional changes. This confirms clusterin and Kim-1 as early and sensitive, noninvasive markers of renal injury. Although Lcn2 did not appear to be specific for kidney toxicity, its rapid response to inflammation and tissue damage in general may suggest its utility in routine toxicity testing.

Mutagenicity of amino acid and glutathione S-conjugates in the Ames test

The mutagenicity of the glutathione S-conjugate S-(1,2-dichlorovinyl)glutathione (DCVG), the cysteine conjugates S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and S-(1,2-dichlorovinyl)-DL-alpha-methylcysteine (DCVMC), and the homocysteine conjugates S-(1,2-dichlorovinyl)-L-homocysteine (DCVHC) and S-(1,2-dichlorovinyl)-DL-alpha-methylhomocysteine (DCVMHC) was investigated in Salmonella typhimurium strain TA2638 with the preincubation assay. DCVC was a strong, direct-acting mutagen; the cysteine conjugate beta-lyase inhibitor aminooxyacetic acid decreased significantly the number of revertants induced by DCVC; rat renal mitochondria (11,000 X g pellet) and cytosol (105,000 X g supernatant) with high beta-lyase activity increased DCVC mutagenicity at high DCVC concentrations. DCVG was also mutagenic without the addition of mammalian activating enzymes; the presence of low gamma-glutamyltransferase activity in bacteria, the reduction of DCVG mutagenicity by aminooxyacetic acid, and the potentiation of DCVG mutagenicity by rat kidney mitochondria and microsomes (105,000 X g pellet) with high gamma-glutamyltransferase activity indicate that gamma-glutamyltransferase and beta-lyase participate in the metabolism of DCVG to mutagenic intermediates. The homocysteine conjugate DCVHC was only weakly mutagenic in the presence of rat renal cytosol, which exhibits considerable gamma-lyase activity, this mutagenic effect was also inhibited by aminooxyacetic acid. The conjugates DCVMC and DCVMHC, which are not metabolized to reactive intermediates, were not mutagenic at concentrations up to 1 mumole/plate. The results demonstrate that gamma-glutamyltransferase and beta-lyase are the key enzymes in the biotransformation of cysteine and glutathione conjugates to reactive intermediates that interact with DNA and thereby cause mutagenicity.

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.

Perturbation of Mitosis through Inhibition of Histone Acetyltransferases: The Key to Ochratoxin A Toxicity and Carcinogenicity?

Ochratoxin A (OTA) is one of the most potent rodent renal carcinogens studied to date. Although controversial results regarding OTA genotoxicity have been published, it is now widely accepted that OTA is not a mutagenic, DNA-reactive carcinogen. Instead, increasing evidence from both in vivo and in vitro studies suggests that OTA may promote genomic instability and tumorigenesis through interference with cell division. The aim of the present study was to provide further support for disruption of mitosis as a key event in OTA toxicity and to understand how OTA mediates these effects. Immortalized human kidney epithelial cells (IHKE) were treated with OTA and monitored by differential interference contrast microscopy for 15 h. Image analysis confirmed that OTA at concentrations ≥ 5 μM, which correlate with plasma concentrations in rats under conditions of carcinogenesis, causes sustained mitotic arrest and exit from mitosis without nuclear or cellular division. Mitotic chromosomes were characterized by aberrant condensation and premature sister chromatid separation associated with altered phosphorylation and acetylation of core histones. To test if OTA directly interferes with histone acetyltransferases (HATs) which regulate lysine acetylation of histones and nonhistone proteins, a cell-free HAT activity assay was conducted using total nuclear extracts of IHKE cells. In this assay, OTA significantly blocked HAT activity in a concentration-dependent manner Overall, results from this study provide further support for a mechanism of OTA carcinogenicity involving interference with the mitotic machinery and suggest HATs as a primary cellular target of OTA.

Identification and Pathway Mapping of Furan Target Proteins Reveal Mitochondrial Energy Production and Redox Regulation as Critical Targets of Furan Toxicity

Furan, a heat-generated food contaminant, is hepatotoxic and carcinogenic in rodents. Furan is oxidized by cytochrome P450 2E1 to cis-2-butene-1,4-dial, a chemically reactive α,β-unsaturated dialdehyde, which has been identified as the key toxic metabolite of furan based on its ability to interact with tissue nucleophiles. In addition to genotoxicity, sustained cytotoxicity mediated through covalent binding of cis-2-butene-1,4-dial to critical target proteins is thought to play a key role in furan carcinogenicity. To identify putative protein targets of reactive furan metabolites, male F344/N rats (n = 5 per dose) were administered a single dose of [3,4-(14)C]-furan (20 mCi/mmol) at doses associated with hepatotoxicity following long-term exposure (0.1 and 2 mg/kg body weight [bw]). Liver proteins were separated by two-dimensional gel electrophoresis and protein spots carrying radiolabel were located by fluorography. In total, 83 discrete protein spots containing (14)C were consistently detected in livers of animals given [3,4-(14)C]-furan at 2.0 mg/kg bw, accounting for 4-5% of the proteome covered by our analyses. Protein spots were excised and digested in gel with trypsin for identification by protein mass spectrometry. Protein database search and subsequent pathway mapping identified 61 proteins localized predominantly in the cytosol and mitochondria, including structural proteins, mitochondrial enzymes involved in glucose metabolism, mitochondrial β-oxidation, and adenosine triphosphate synthesis, and proteins that participate in the maintenance of redox homeostasis and protein folding. Collectively, our data suggest that functional loss of several individual proteins and interference with pathways, most notably mitochondrial energy production, redox regulation, and protein folding, may combine to disrupt cell homeostasis and cause hepatocyte cell death.

Specificity of aminoacylase III-mediated deacetylation of mercapturic acids

Trichloroethylene (TCE) and other halogenated alkenes are known environmental contaminants with cytotoxic and nephrotoxic effects, and are potential carcinogens. Their metabolism via the mercapturate metabolic pathway was shown to lead to their detoxification. The final products of this pathway, mercapturic acids or N-acetyl-l-cysteine S-conjugates, are secreted into the lumen in the renal proximal tubule. The proximal tubule may also deacetylate mercapturic acids, and the resulting cysteine S-conjugates are transformed by cysteine S-conjugate β-lyases to nephrotoxic reactive thiols. The specificity and rate of mercapturic acid deacetylation may determine the toxicity of certain mercapturic acids; however, the exact enzymologic processes involved are not known in detail. In the present study we characterized the kinetics of the recently cloned mouse aminoacylase III (AAIII) toward a wide spectrum of halogenated mercapturic acids and N-acetylated amino acids. In general, the Vmax value of AAIII was significantly larger with chlorinated and brominated mercapturic acids, whereas fluorination significantly decreased it. The enzyme deacetylated mercapturic acids derived from the TCE metabolism including N-acetyl-S-(1,2-dichlorovinyl)-l-cysteine (NA-1,2-DCVC) and N-acetyl-S-(2,2-dichlorovinyl)-l-cysteine (NA-2,2-DCVC). Both mercapturic acids induced cytotoxicity in mouse proximal tubule mPCT cells expressing AAIII, which was decreased by an inhibitor of β-lyase, aminooxyacetate. The toxic effect of NA-2,2-DCVC was smaller than that of NA-1,2-DCVC, indicating that factors other than the intracellular activity of AAIII mediate the cytotoxicity of these mercapturic acids. Our results indicate that in proximal tubule cells, AAIII plays an important role in deacetylating several halogenated mercapturic acids, and this process may be involved in their cyto- and nephrotoxicity.

Biotransformation of 2,3,3,3-tetrafluoropropene (HFO-1234yf)

2,3,3,3-Tetrafluoropropene (HFO-1234yf) is a non-ozone-depleting fluorocarbon replacement with a low global warming potential which has been developed as refrigerant. The biotransformation of HFO-1234yf was investigated after inhalation exposure. Male Sprague-Dawley rats were exposed to air containing 2000, 10,000, or 50,000 ppm HFO-1234yf for 6 h and male B6C3F1 mice were exposed to 50,000 ppm HFO-1234yf for 3.5 h in a dynamic exposure chamber (n=5/concentration). After the end of the exposure, animals were individually housed in metabolic cages and urines were collected at 6 or 12-hour intervals for 48 h. For metabolite identification, urine samples were analyzed by (1)H-coupled and decoupled (19)F-NMR and by LC/MS-MS or GC/MS. Metabolites were identified by (19)F-NMR chemical shifts, signal multiplicity, (1)H-(19)F coupling constants and by comparison with synthetic reference compounds. In all urine samples, the predominant metabolites were two diastereomers of N-acetyl-S-(3,3,3-trifluoro-2-hydroxy-propyl)-l-cysteine. In (19)F-NMR, the signal intensity of these metabolites represented more than 85% (50,000 ppm) of total (19)F related signals in the urine samples. Trifluoroacetic acid, 3,3,3-trifluorolactic acid, 3,3,3-trifluoro-1-hydroxyacetone, 3,3,3-trifluoroacetone and 3,3,3-trifluoro-1,2-dihydroxypropane were present as minor metabolites. Quantification of N-acetyl-S-(3,3,3-trifluoro-2-hydroxy-propyl)-l-cysteine by LC/MS-MS showed that most of this metabolite (90%) was excreted within 18 h after the end of exposure (t(1/2) app. 6 h). In rats, the recovery of N-acetyl-S-(3,3,3-trifluoro-2-hydroxy-propyl)-l-cysteine excreted within 48 h in urine was determined as 0.30+/-0.03, 0.63+/-0.16, and 2.43+/-0.86 micromol at 2000, 10,000 and 50,000 ppm, respectively suggesting only a low extent (<<1% of dose received) of biotransformation of HFO-1234yf. In mice, the recovery of this metabolite was 1.774+/-0.4 mumol. Metabolites identified after in vitro incubations of HFO-1234yf in liver microsomes from rat, rabbit, and human support the metabolic pathways of HFO-1234yf revealed in vivo. The obtained results suggest that HFO-1234yf is subjected to a typical biotransformation reaction for haloolefins, likely by a cytochrome P450 2E1-catalyzed formation of 2,3,3,3-tetrafluoroepoxypropane at low rates, followed by glutathione conjugation or hydrolytic ring opening.

Biosynthesis of toxic glutathione conjugates from halogenated alkenes

Glutathione conjugation has been identified as an important detoxication reaction. However, several glutathione-dependent bioactivation reactions have been identified. Current knowledge on the mechanisms and the possible biological importance of these reactions is discussed in this article. Several polychlorinated alkenes are bioactivated in a complex, glutathione-dependent pathway. The first step is hepatic glutathione S-conjugate formation followed by cleavage to the corresponding cysteine S-conjugates, and, after translocation to the kidney, metabolism by renal cysteine conjugate beta-lyase. Beta-lyase-dependent metabolism of halovinyl cysteine S-conjugates yields electrophilic thioketenes, whose covalent binding to cellular macromolecules is likely responsible for the observed nephrotoxicity of the parent compounds.