Edward A. Lock

Biological Qualification of Biomarkers of Chemical-Induced Renal Toxicity in Two Strains of Male Rat

This study reports the evaluation of four urinary biomarkers of renal toxicity, α-glutathione-S-transferase (α-GST), μ-GST, clusterin, and renal papillary antigen-1 (RPA-1), in male Sprague-Dawley and Han-Wistar rats given cisplatin, gentamicin, or N-phenylanthranilic acid (NPAA). Kidney injury was diagnosed histopathologically, according to site/nature of renal injury, and graded for severity. The area under the receiver operating characteristic (ROC) curve was used to compare the diagnostic accuracy of each exploratory renal biomarker with traditional indicators of renal function and injury (blood urea nitrogen [BUN], serum creatinine [sCr] as well as urinary N-acetyl-β-D-glucosaminidase [NAG] and protein). These analyses showed that increased urinary α-GST was superior to BUN, sCr, and NAG for diagnosis of proximal tubular (PT) degeneration/necrosis. Paradoxically, urinary α-GST was decreased in the presence of collecting duct (CD) injury without PT injury (NPAA administration). RPA-1 demonstrated high specificity for CD injury, superior to all of the reference biomarkers. The clusterin response correlated well with tubular injury, whatever the location, particularly when regeneration was present (superior to all of the reference markers for cortical tubular regeneration). There was no conclusive evidence for the diagnostic utility of μ-GST. The data were submitted for qualification review by the European Medicines Agency and the US Food and Drug Administration. Both agencies concluded that the data justified the qualification of RPA-1 and increased the level of evidence for, and clarified the context of use of, the previously qualified clusterin for use in male rats. These biomarkers can be used in conjunction with traditional clinical chemistry markers and histopathology in Good Laboratory Practice rodent toxicology studies used to support renal safety studies in clinical trials. Qualification of α-GST must await further explanation of the differences in response to PT and CD injury.

Immunocytochemical localization of cytochrome P-450 in hepatic and extra-hepatic tissues of the rat with a monoclonal antibody against cytochrome P-450 c

The cellular distribution of cytochrome P-450 has been studied in the liver and a number of extrahepatic tissues in the rat by immunocytochemistry, using an antibody raised against cytochrome P-450 form c. Immunoreactive cytochrome P-450, most probably form c, was found in the proximal tubules of the kidney, in the Clara cells of the lung, and in the olfactory epithelium and Bowmans glands of the olfactory tissue, in addition to its location in the liver. Immunoreactive cytochrome P-450 was not found in the small intestine, the testes or the adrenal gland, although these organs are known to contain isoenzymes of cytochrome P-450. The use of antibody titration enabled the effects of phenobarbitone, beta-naphthoflavone and clofibrate on the content and distribution of immunoreactive cytochrome P-450 to be studied in both the liver and in the other organs discussed. Phenobarbitone induces epitope-specific cytochrome P-450 in the centrilobular cells of the liver but has no effect in any of the other tissues studied. Clofibrate is without effect on the levels of immunoreactive cytochrome P-450 in any of the tissues studied. In contrast, beta-naphthoflavone induces immunoreactive cytochrome P-450 in the periportal region of the liver, and also in the Clara cells of the lung, in the enterocytes of the small intestine and in the proximal tubules of the kidney. Of all of the tissues studied, in which immunoreactive cytochrome P-450 could be detected, only the olfactory epithelium failed to undergo enzyme induction following treatment with beta-naphthoflavone.

The carcinoGENOMICS project: Critical selection of model compounds for the development of omics-based in vitro carcinogenicity screening assays

Recent changes in the European legislation of chemical-related substances have forced the scientific community to speed up the search for alternative methods that could partly or fully replace animal experimentation. The Sixth Framework Program project carcinoGENOMICS was specifically raised to develop omics-based in vitro screens for testing the carcinogenic potential of chemical compounds in a pan-European context. This paper provides an in-depth analysis of the complexity of choosing suitable reference compounds used for creating and fine-tuning the in vitro carcinogenicity assays. First, a number of solid criteria for the selection of the model compounds are defined. Secondly, the strategy followed, including resources consulted, is described and the selected compounds are briefly illustrated. Finally, limitations and problems encountered during the selection procedure are discussed. Since selecting an appropriate set of chemicals is a frequent impediment in the early stages of similar research projects, the information provided in this paper might be extremely valuable.

Solvents and Parkinson disease: A systematic review of toxicological and epidemiological evidence

Parkinson disease (PD) is a debilitating neurodegenerative motor disorder, with its motor symptoms largely attributable to loss of dopaminergic neurons in the substantia nigra. The causes of PD remain poorly understood, although environmental toxicants may play etiologic roles. Solvents are widespread neurotoxicants present in the workplace and ambient environment. Case reports of parkinsonism, including PD, have been associated with exposures to various solvents, most notably trichloroethylene (TCE). Animal toxicology studies have been conducted on various organic solvents, with some, including TCE, demonstrating potential for inducing nigral system damage. However, a confirmed animal model of solvent-induced PD has not been developed. Numerous epidemiologic studies have investigated potential links between solvents and PD, yielding mostly null or weak associations. An exception is a recent study of twins indicating possible etiologic relations with TCE and other chlorinated solvents, although findings were based on small numbers, and dose-response gradients were not observed. At present, there is no consistent evidence from either the toxicological or epidemiologic perspective that any specific solvent or class of solvents is a cause of PD. Future toxicological research that addresses mechanisms of nigral damage from TCE and its metabolites, with exposure routes and doses relevant to human exposures, is recommended. Improvements in epidemiologic research, especially with regard to quantitative characterization of long-term exposures to specific solvents, are needed to advance scientific knowledge on this topic.

Oxidative stress induced by potassium bromate exposure results in altered tight junction protein expression in renal proximal tubule cells

Potassium bromate (KBrO3) is an oxidising agent that has been widely used in the food and cosmetic industries. It has shown to be both a nephrotoxin and a renal carcinogen in in vivo and in vitro models. Here, we investigated the effects of KBrO3 in the human and rat proximal tubular cell lines RPTEC/TERT1 and NRK-52E. A genome-wide transcriptomic screen was carried out from cells exposed to a sub-lethal concentration of KBrO3 for 6, 24 and 72xa0h. Pathway analysis identified “glutathione metabolism”, “Nrf2-mediated oxidative stress” and “tight junction (TJ) signalling” as the most enriched pathways. TJ signalling was less impacted in the rat model, and further studies revealed low transepithelial electrical resistance (TEER) and an absence of several TJ proteins in NRK-52E cells. In RPTEC/TERT1 cells, KBrO3 exposure caused a decrease in TEER and resulted in altered expression of several TJ proteins. N-Acetylcysteine co-incubation prevented these effects. These results demonstrate that oxidative stress has, in conjunction with the activation of the cytoprotective Nrf2 pathway, a dramatic effect on the expression of tight junction proteins. The further understanding of the cross-talk between these two pathways could have major implications for epithelial repair, carcinogenesis and metastasis.

Transcriptomic alterations induced by Ochratoxin A in rat and human renal proximal tubular in vitro models and comparison to a rat in vivo model

Ochratoxin A (OTA) is a widely studied compound due to its role in renal toxicity and carcinogenicity. However, there is still no consensus on the exact mechanisms of toxicity or carcinogenicity. In the current study, we analysed the effect of OTA on three human renal proximal tubular models (human primary, RPTEC/TERT1 and HK-2 cells) and two rat renal proximal tubular models (rat primary and NRK-52E cells). Global transcriptomics analysis at two exposure times was performed to generate a set of 756 OTA sensitive genes. This gene set was then compared in more detail across all models and additionally to a rat in vivo renal cortex model. The results demonstrate a well-conserved response across all models. OTA resulted in deregulation of a number of pathways including cytoskeleton, nucleosome regulation, translation, transcription, ubiquitination and cell cycle pathways. Interestingly, the oxidative stress activated Nrf2 pathway was not enriched. These results point to an epigenetic action of OTA, perhaps initiated by actin binding as the actin remodelling gene, advillin was the highest up-regulated in all models. The largest model differences were observed between the human and the rat in vitro models. However, since the human in vitro models were more similar to the rat in vivo model, it is more likely that these differences are model-specific rather than species-specific per se. This study demonstrates the usefulness of in vitro cell culture models combined with transcriptomic analysis for the investigation of mechanisms of toxicity and carcinogenicity. In addition, these results provide further evidence supporting a non-genotoxic mechanism of OTA-induced carcinogenicity.

Effect of ascorbic acid, acivicin and probenecid on the nephrotoxicity of 4-aminophenol in the Fischer 344 rat.

Abstract4-Aminophenol (p-aminophenol, PAP) causes selective necrosis to the pars recta of the proximal tubule in Fischer 344 rats. The basis for this selective toxicity is not known but PAP can undergo oxidation in a variety of systems to form the 4-aminophenoxy free radical. Oxidation or disproportionation of this radical will form 1,4-benzoquinoneimine which can covalently bind to cellular macromolecules. We have recently reported that a glutathione conjugate of PAP, 4-amino-3-S-glutathionylphenol, is more toxic to the kidney than the parent compound itself. In this study we have examined the distribution and covalent binding of radiolabel from 4-[ring3H]-aminophenol in the plasma, kidney and liver of rats 24 h after dosing and related these findings to the extent of nephrotoxicity. In addition, we have examined the effect of ascorbic acid which will slow the oxidation of PAP; acivicin, an inhibitor of γ-glutamyltransferase and hence the processing of glutathione-derived conjugates; and probenecid, an inhibitor of organic anion transport on the nephrotoxicity produced by PAP. Administration of a single dose of PAP at 458 or 687 μmol kg−1 produced a dose-related alteration in renal function within 24 h which was associated with proximal tubular necrosis. The lesion at the lower dose was restricted to the S3 proximal tubules in the medullary rays, while at the higher dose it additionally affected the S3 tubules in the pars recta region of the cortex. Administration of ascorbic acid protected rats against the nephrotoxicity produced by PAP, markedly reducing the effect on renal function, and the extent of renal tubular necrosis. Associated with this protection was a reduction in the concentration of both total and covalently bound radiolabel from PAP in the kidney. In contrast, prior treatment with acivicin slightly potentiated the nephrotoxicity of PAP at the lower dose of 458 μmol kg−1, by increasing the extent of proximal tubular necrosis and azotemia. In association with this potentiation the concentration of both total and covalently bound radiolabel from PAP in the kidney was increased. Prior treatment with probenecid had little or no effect on the nephrotoxicity of PAP or on the distribution of radiolabel from PAP in the kidney. These studies indicate that oxidation of PAP to form a metabolite which can covalently bind to renal proteins may be an important step in the nephrotoxic process and that treatment with ascorbic acid reduces this and thereby affords protection.

Sensitive and early markers of renal injury: where are we and what is the way forward?

Measurement of total urinary protein, glucose, and the appearance of certain enzymes such as N-acetyl-b-(D)glucosaminidase has been used in both animals and man for many years to provide insight into the onset of renal injury. However, it is clear that serum creatinine and such markers are not very sensitive and are generally only raised when acute renal injury or chronic renal injury is well established. With the coming of the ‘‘omics’’ age and the development of transcriptomics, proteomics, and metabonomics, it has become possible to interrogate urine samples to identify new proteins or small organic molecules appearing in urine as the result of a toxic insult or in models of renal disease. This new technology has stimulated considerable interest and effort in trying to identify novel, sensitive, and early urinary biomarkers of renal injury. A sensitive tissue marker of tubular injury, which can be used to identify or confirm the presence of epithelial cell injury even when morphological changes are minimal during the development of a new drug or pesticide or for the evaluation of biopsy material, would be welcomed by all concerned. However, history tells us that it is not one single urinary measurement, but a number of measurements that indicate that renal injury has occurred. Thus, ideally, we are looking for a number of biomarkers that together may inform us not only of the presence of injury but perhaps in which part of the nephron the injury is located.

Bone marrow and renal injury associated with haloalkene cysteine conjugates in calves

Abstractu2002Almost 40 years ago, it was reported that cattle-feed which had been extracted with hot trichloroethylene and then fed to calves produced renal injury and a fatal aplastic anaemia. The toxic factor was subsequently identified as S-(1,2-dichlorovinyl)-L-cysteine (DCVC). These original findings have been confirmed, a single intravenous dose of DCVC at 4u2009mg/kg, or 0.4u2009mg/kg intravenously per day administered for 10 days to calves produced aplastic anaemia, and renal injury after a single dose of 4u2005mg/kg. The toxicity to calves of a number of other haloalkene cysteine conjugates has been examined to ascertain whether, like DCVC, they produce bone marrow and renal injury. Intravenous administration of the N-acetyl cysteine conjugate of DCVC produced renal but not bone marrow injury at a molar equivalent dose to DCVC, indicating that the calf can deacetylate the mercapturic acid and further that sufficient chemical had reached the kidney to be a substrate for the enzyme cysteine conjugate β-lyase. However, intravenous administration of the α-methyl analogue of DCVC, which cannot undergo metabolism via the enzyme cysteine conjugate β-lyase, was without toxicity at doses about five-fold higher than DCVC. These latter findings provide strong evidence that metabolism of DCVC via the enzyme β-lyase is necessary for bone marrow and renal injury to occur. The cysteine conjugates of perchloro ethylene and hexachloro-1,3-butadiene(HCBD) when given intravenously to calves at molar equivalent doses to DCVC, or above, did not produce either bone marrow or renal injury. In contrast, intravenous administration of the cysteine conjugate of tetrafluoroethylene (TFEC) produced severe renal tubular injury in calves without affecting the bone marrow. In vitro studies with these haloalkene cysteine conjugates showed, like DCVC, that they were good substrates for calf renal cysteine conjugate β-lyase and toxic to renal cells as judged by their ability to reduce organic anion and cation transport by slices of calf renal cortex and inhibit the renal enzyme glutathione reductase. Calves were also dosed either orally or intravenously with HCBD to assess its toxicity. HCBD at higher molar equivalent doses than DCVC produced mid-zonal necrosis in the liver, renal tubular necrosis but no bone marrow injury in calves. The key findings emerging from these studies are (1) that none of the other cysteine conjugates, at molar equivalent doses to DCVC and above, produce bone marrow injury in calves, (2) TFEC produced only renal injury, suggesting that sufficient of the other conjugates had not reached the kidney for metabolism by β-lyase to produce cytotoxicity and (3) that HCBD itself is more toxic than its cysteine or mercapturic acid conjugate, suggesting that pharmacokinetics and disposition are important factors in determining the toxicity of these conjugates to calves. Further studies are needed to understand the basis for the selective toxicity of DCVC to the bone marrow of calves.

Nephrotoxicity of 4-amino-3-S-glutathionylphenol and its modulation by metabolism or transport inhibitors

The nephrotoxicity of 4-amino-3-S-glutathionylphenol (PAP-GSH), a known metabolite of 4-aminophenol (PAP), was determined in male Fischer 344 rats. Administration of a single dose of 40 or 60 μmol kg−1 caused a marked elevation in blood urea nitrogen and an increase in the urinary excretion of glucose, protein and γ-glutamyltransferase (GGT). These changes were associated with histological alterations in the proximal tubule, where at the lower dose the lesion was restricted to the S3 region of the proximal tubule in the medullary rays, while at the higher dose the lesion extended to affect the S3 region in both the medullary rays and the outer stripe of the outer medulla. Studies with [35S]-PAP-GSH at 40 μmol kg−1 showed selective retention of radioactivity in the kidney, relative to other organs 24 h after dosing and that some radioactivity was covalently bound to renal proteins. Pretreatment of animals with probenecid, an inhibitor of renal organic anion transport, or aminooxyacetic acid, an inhibitor of cysteine conjugate ß-lyase, had little or no effect on the toxicity. In contrast, pretreatment of animals with acivicin, an inhibitor of γ-glutamyltransferase, or co-administration of PAP-GSH with ascorbic acid almost completely protected against the nephrotoxicity. This protection was associated with a decreased concentration of radioactivity from [35S]-PAP-GSH in the kidneys and a decrease in the amount covalently bound to renal protein. Thus, the nephrotoxicity of PAP-GSH may be mediated by oxidation and further processing of the glutathione conjugate via γ-glutamyltransferase.