Inter-laboratory study of human in vitro toxicogenomics-based tests as alternative methods for evaluating chemical carcinogenicity: a bioinformatics perspective.

The assessment of the carcinogenic potential of chemicals with alternative, human-based in vitro systems has become a major goal of toxicogenomics. The central read-out of these assays is the transcriptome, and while many studies exist that explored the gene expression responses of such systems, reports on robustness and reproducibility, when testing them independently in different laboratories, are still uncommon. Furthermore, there is limited knowledge about variability induced by the data analysis protocols. We have conducted an inter-laboratory study for testing chemical carcinogenicity evaluating two human in vitro assays: hepatoma-derived cells and hTERT-immortalized renal proximal tubule epithelial cells, representing liver and kidney as major target organs. Cellular systems were initially challenged with thirty compounds, genome-wide gene expression was measured with microarrays, and hazard classifiers were built from this training set. Subsequently, each system was independently established in three different laboratories, and gene expression measurements were conducted using anonymized compounds. Data analysis was performed independently by two separate groups applying different protocols for the assessment of inter-laboratory reproducibility and for the prediction of carcinogenic hazard. As a result, both workflows came to very similar conclusions with respect to (1) identification of experimental outliers, (2) overall assessment of robustness and inter-laboratory reproducibility and (3) re-classification of the unknown compounds to the respective toxicity classes. In summary, the developed bioinformatics workflows deliver accurate measures for inter-laboratory comparison studies, and the study can be used as guidance for validation of future carcinogenicity assays in order to implement testing of human in vitro alternatives to animal testing.

Asymptomatic Corneal Keratopathy Secondary to Hypertyrosinaemia Following Low Dose Nitisinone and a Literature Review of Tyrosine Keratopathy in Alkaptonuria.

Nitisinone, although unapproved for use in alkaptonuria (AKU), is currently the only homogentisic acid lowering therapy with a potential to modify disease progression in AKU. Therefore, safe use of nitisinone off-label requires identifying and managing tyrosine keratopathy. A 22-year-old male with AKU commenced 2 mg daily nitisinone after full assessment. He was issued an alert card explaining potential ocular symptoms such as red eye, tearing, ocular pain and visual impairment and how to manage them. On his first and second annual follow-up visits to the National Alkaptonuria Centre (NAC), there was no corneal keratopathy on slit lamp examination. On his third follow-up annual visit to the NAC, he was found to have typical dendritiform corneal keratopathy in both eyes which was asymptomatic. Nitisinone was suspended until a repeat slit lamp examination, 2 weeks later, confirmed that the keratopathy had resolved. He recommenced nitisinone 2 mg daily with a stricter low protein diet. On his fourth annual follow-up visit to the NAC, a routine slit lamp examination showed mild corneal keratopathy in the left eye. This is despite him reporting no ocular symptoms. This case highlights the fact that corneal keratopathy can occur without symptoms and any monitoring plan with off-label use of nitisinone in AKU will need to take this possibility into account. This is also the first time that typical corneal keratopathy has been described with the use of low dose nitisinone in AKU without symptoms.

Sodium benzoate attenuates D-serine induced nephrotoxicity in the rat.

D-Serine causes selective necrosis to the straight portion of the rat renal proximal tubules. The onset is rapid, occurring within 3-4 h and accompanied by proteinuria, glucosuria and aminoaciduria. The metabolism of D-serine by D-amino acid oxidase (D-AAO) may be involved in the mechanism of toxicity. D-AAO is localized within the peroxisomes of renal tubular epithelial cells, which is also the location of D-serine reabsorption. To address the role of D-AAO in D-serine-induced nephrotoxicity, we have examined the effect of sodium benzoate (SB) on the renal injury. SB has been shown to be a potent, competitive inhibitor of kidney D-AAO in vitro. Male Alderley Park rats were exposed to D-serine (500 mg/kg i.p.) 1 h after exposure to SB (125, 250, 500 or 750 mg/kg i.p.). Urine was collected for 0-6 h, then terminal plasma samples and kidneys were taken at 6.5 h. A second group of animals was given SB (500 mg/kg) followed by D-serine (500 mg/kg i.p.; 1 h later) and urine was collected after 0-6, 6-24 and 24-48 h. Terminal plasma samples and kidneys were taken at 48 h. 1H NMR spectroscopic analysis of urine, combined with principal component analysis, demonstrated that SB was able to prevent D-serine-induced perturbations to the urinary profile in a dose dependent manner. This was confirmed by measurement of plasma creatinine and urinary glucose and protein and histopathological examination of the kidneys. Assessment 48 h after D-serine administration revealed that nephrotoxicity was observed in animals pre-treated with SB (500 mg/kg) although the extent of injury was less pronounced than following D-serine alone. These results demonstrate that whilst prior exposure to SB prevents the initial onset of D-serine-induced nephrotoxicity, renal injury is still apparent at later time points. D-AAO activity in the kidney was decreased by 50% 1 h after dosing with SB suggesting that inhibition of this enzyme may be responsible for the observed protection.

D-Serine-induced nephrotoxicity: a HPLC-TOF/MS-based metabonomics approach.

HPLC-MS-based metabonomic analysis was used to investigate urinary metabolic perturbations associated with D-serine-induced nephrotoxicity. D-Serine causes selective necrosis of the proximal straight tubules in the rat kidney accompanied by aminoaciduria, proteinuria and glucosuria. Alderely Park (Wistar-derived) rats were dosed with either D-serine (250 mg/kg ip) or vehicle (deionised water) and urine was collected at 0-12, 12-24, 24-36 and 36-48 h post-dosing. Samples were analysed using a Waters Alliance HT 2795 HPLC system coupled to a Waters Micromass Q-ToF-micro equipped with an electrospray source operating in either positive or negative ion mode. Changes to the urinary profile were detected at all time points compared to control. In negative ion mode, increases were observed in serine (m/z=103.0077), m/z=104.0376 (proposed to be hydroxypyruvate) and glycerate (m/z=105.0215), the latter being metabolites of D-serine. Furthermore, an increase in tryptophan, phenylalanine and lactate and decreases in methylsuccinic acid and sebacic acid were observed. Positive ion analysis revealed a decrease in xanthurenic acid, which has previously been assigned and reported using HPLC-MS following exposure to mercuric chloride and cyclosporine A. A general aminoaciduria, including proline, methionine, leucine, tyrosine and valine was also observed as well as an increase in acetyl carnitine. Investigation of additional metabolites altered as a result of exposure to D-serine is on-going. Thus, HPLC-MS-based metabonomic analysis has provided information concerning the mechanism of D-serine-induced renal injury.

Neuropathological studies on cycloate-induced neuronal cell death in the rat brain.

The herbicide cycloate (carbamothioic acid, ethyl(cyclohexyl)-S-ethyl ester) given as a single oral dose to rats, caused selective neuronal cell death in two regions in the rat forebrain, the pyramidal neurons of layers II-III throughout the pyriform cortex and in granule cells of the caudal ventro-lateral dentate gyrus. Male Alderley Park rats, 6-8-week-old, were given a single oral dose of either 0 or 2000 mg/kg cycloate and killed for neuropathological investigation 1, 2, 3, 7, 14 or 28 days after dosing, using a regime of perfusion fixation with modified Karnovsky's fixative, followed by routine paraffin embedding. Seven transverse levels of brain were examined from each rat. Cycloate-induced neuronal cell death was seen in the pyriform cortex 1 day after dosing and persisted through to Day 28, the lesion was more marked in the rostral compared to the caudal region of the pyriform cortex. Neuronal cell death was also observed in the ventro-lateral caudal dentate gyrus on Days 1-14, day after dosing. In the early stages, Days 1-3 and to a lesser extent Day 7, the neuronal cell death resembled apoptosis, characterized by condensation of nuclear material, cell shrinkage and strong cytoplasmic eosinophilia. By Days 14 and 28 and to a lesser extent Day 7, the cell death resembled necrosis, i.e. karyorrhectic nuclei with pale irregular cytoplasm. Microglial accumulation was associated with the neuronal cell injury. In control brains, an occasional apoptotic body was seen in both the pyriform cortex and dentate gyrus. Our results demonstrate that cycloate is a novel neurotoxicant, which following a single large oral dose induces a cell specific and highly localized forebrain lesion. The time course data analyzed temporally, suggests that cycloate may cause an up regulation of apoptosis in selected regions of the adult brain.

Characterisation of kainate receptor mediated whole-cell currents in rat cultured cerebellar granule cells.

Whole-cell voltage clamp recordings have been used to identify and characterise inward currents mediated by native kainate receptors in rat cultured cerebellar granule cells. While the selective AMPA receptor antagonist GYKI 53655 (50 microM) completely abolished inward currents evoked by AMPA (10-100 microM) in the presence of cyclothiazide (100 microM), kainate evoked currents in cells pretreated with concanavalin A (Con A) always showed a component (35-140 pA, n = 13) resistant to blockade. The majority (73+/-7%, n = 5) of GYKI 53655-resistant kainate-evoked inward currents remained in the presence of 100 microM AMPA. However, these currents were reversibly blocked by the competitive AMPA/kainate receptor antagonist NBQX (100 microM). (2S, 4R)-4-methylglutamate (SYM 2081, 10 microM) evoked inward currents in Con A treated cells (15-60 pA, n = 7), which were resistant to complete blockade by GYKI 53655 (50 microM) but antagonised by NBQX (100 microM). Kainate-evoked responses in the presence of GYKI 53655 (50 microM) had linear or slightly outwardly rectifying current-voltage (I-V) relationships in all cells examined (n = 5) and were resistant to blockade by Joro spider toxin (JsTx, 1 microM; n = 5). These results provide evidence that rat cultured cerebellar granule cells express functional kainate receptors made up of subunits which are edited at the Q/R site, and that SYM 2081 is an agonist at these native kainate receptors with a greater selectivity than kainate itself.

Possible role of nitric oxide in the development of L-2-chloropropionic acid-induced cerebellar granule cell necrosis.

1. L-2-Chloropropionic acid (L-CPA) produces selective neuronal cell necrosis in rat cerebellum when administered orally at 750 mg kg-1 that is mediated in part through activation of N-methyl-D-aspartate (NMDA) receptors. Cerebellar granule cell death occurs between 30 and 36 h following L-CPA administration exhibiting a number of features in common with excitatory amino acid-induced cell death. We have used this in vivo model to examine the neurochemical processes following L-CPA-induced activation of NMDA receptors leading to neuronal cell death in the rat cerebellum. 2. The effects of a number of compounds which potently block nitric oxide synthase in vitro were examined on L-CPA-induced neurotoxicity 48 h following L-CPA dosing, to discover whether the neuronal cell death is mediated in part by excessive nitric oxide generation. Four inhibitors were studied, NG-nitro-L-arginine (L-NOARG), NG-nitro-L-arginine methyl ester (L-NAME), NG-iminoethyl-L-ornithine (L-NIO) and 3-bromo-7-nitroindazole (BrNI). 3. L-NAME (50 mg kg-1, i.p. twice daily) and BrIN (50 mg kg-1, i.p. twice daily) administration prevented the L-CPA-induced loss of granule cells which can reach up to 80-90% of the total cell number in rats treated with L-CPA alone. L-NOARG (50 mg kg-1, i.p. twice daily) and L-NIO administered at either 25 or 100 mg kg-1, twice daily did not produce any significant protection against L-CPA-induced neurotoxicity. 4. Both L-NAME and BrIN also prevented the L-CPA-induced increase in cerebellar water content and sodium concentrations. L-NIO when administered at the highest doses prevented the increase in cerebellar sodium concentration but not water content. L-NIO and L-NOARG were ineffective in preventing the L-CPA-induced increases in cerebellar water and sodium concentrations. 5. L-CPA-induced reductions in cerebellar aspartate and glutamate concentrations and increases in glutamine and GABA concentrations were prevented by L-NAME and BrIn, but not by L-NIO or L-NOARG. Also reductions in L-[3H]-glutamate binding to glutamate ionotrophic and metabotrophic receptors in the granule cell layer of rat cerebellum was prevented by L-NAME and BrIN, but not L-NIO or L-NOARG. 6. In conclusion, the neuroprotection offered by L-NAME and BrIN suggests that L-CPA-induced cerebellar granule cell necrosis is possibly mediated by or associated with excessive generation of nitric oxide. The inability of nitric oxide synthase inhibitors, L-NOARG and L-NIO to afford protection may result from their limited penetration into the brain (L-NIO) or rapid dissociation from the enzyme.

Regulation of rat olfactory glutathione S-transferase expression. Investigation of sex differences, induction, and ontogenesis.

The glutathione S-transferases (GSTs) of rat olfactory epithelium have been characterised with regard to sex differences, induction, and developmental regulation, and compared to those of the liver. Olfactory cytosolic GST activity with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate was similar in both male and female animals, and there were no differences in subunit profile. Administration of trans-stilbene oxide and beta-naphthoflavone had no effect on olfactory GST activity with CDNB, although phenobarbitone treatment resulted in a small, but significant, increase in activity (130% compared to controls). HPLC analysis of subunit profiles indicated that all three agents induced olfactory subunit 1b and decreased subunit 6. The effect of age (3 to 84 days) on both cytosolic and microsomal CDNB activity was examined. In the liver, cytosolic activity was low at 3 days and climbed steadily to reach maximal levels around 28 days, but microsomal activity was relatively constant at all ages. Olfactory cytosolic activity was similar at all ages; microsomal activity was low until 21 days and then increased to reach a maximum at 56 days. Changes in individual cytosolic subunits were assessed by SDS-PAGE followed by immunoblotting. The significance of these results with regard to putative physiological roles for olfactory GSTs is discussed.

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 Bowman's 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.

A monoclonal antibody raised to rat liver cytochrome P-448 (form C) which recognises an epitope common to many other forms of cytochrome P-450.

A murine monoclonal antibody has been raised against a partially purified preparation of hepatic cytochrome P-448 (form c) from beta-naphthoflavone-treated rats. The monoclonal origin of the antibody was established by limiting dilution culture and isoelectricfocusing. The antibody has been designated 3/4/2. It reacts with apparently homogeneous cytochrome P-448 from rat liver in solid phase assay. It also cross reacts with a number of other cytochromes P-450, from rat and rabbit. In addition, a positive reaction was obtained with microsomal fractions from a variety of species, including man. None of the species tested was negative. The antibody does not react appreciably with purified haemoproteins other than cytochromes P-450. Antibody 3/4/2 is not inhibitory, either in reconstituted systems or with intact microsomal fraction. However, evidence was obtained that the antibody does cause some perturbation of the tertiary structure of the apoprotein at or near the haem.

[125I]endothelin binding in rat cerebellum is increased following L-2-chloropropionic-acid-induced granule cell necrosis.

The systemic administration of L-2-chloropropionic acid (L-CPA) to rats produced a marked depletion of cerebellar granule cells (> 80% of the total) when administered in a single oral dose of 750 or 250 mg/kg/day for 3 days. The nature of the L-CPA-induced neurotoxicity is currently unknown but it exhibits a number of features in common with excitatory amino acid-induced neuronal cell death. We observed an increase in [125I]endothelin-1 (ET-1) binding in the cerebellar cortex, as measured by quantitative receptor autoradiography, which occurs at 48 h, but not 24 h, following the 750 mg/kg L-CPA dosing regimen. The increase in [125I]ET-1 binding did not parallel the cellular damage and resultant astrocyte proliferation, as measured by GFAP immunoreactivity, which was primarily confined to the granular layer of the cerebellar cortex. The increased [125I]ET-1 binding occurred in the molecular layer of the cerebellar cortex (controls 2.03 +/- 0.26 fmol/mg tissue; L-CPA-treated 6.69 +/- 0.45 fmol/mg tissue, n = 6; p < 0.01, Student's t-test) which appeared to contain astrocytic processes originating from the large increase in astrocyte number situated in the granular layer. Pretreatment of the rats with the irreversible NMDA receptor antagonist, MK-801, protected the cerebellar granule cells against the L-CPA neurotoxicity and also prevented the increase in [125I]ET-1 binding in the cerebellar cortex. The increased [125I]ET-1 binding in rat cerebellum appears to be linked to the reactive gliosis that occurs in association with neuronal cell injury.

Symposium overview: the role of glutathione in neuroprotection and neurotoxicity.

Although the cytoprotective effects of glutathione (GSH) are well established, additional roles for GSH in brain function are being identified that provide a pharmacological basis for the relationship between alterations in GSH homeostasis and the development of certain neurodegenerative processes. Thus, GSH and glutathione disulfide (GSSG) appear to play important functional roles in the central nervous system (CNS). A symposium, focussing on the emerging science of the roles of GSH in the brain, was held at the 37th annual meeting of the Society of Toxicology, with the emphasis on the role of glutathione in neuroprotection and neurotoxicity. Jean Francois Ghersi-Egea opened the symposium by describing the advances in our understanding of the role of the blood-brain and blood-cerebral spinal fluid (CSF) barriers in either limiting or facilitating the access of xenobiotics into the brain. Once within the brain, a multitude of factors will determine whether a chemical causes toxicity and at which sites such toxicity will occur. In this respect, it is becoming increasingly clear that GSH and its various conjugation enzymes are not evenly distributed throughout the brain. Martin Philbert discussed how this regional heterogeneity might provide a potential basis for the theory of differential sensitivity to neurotoxicants, in various regions of the brain. For certain chemicals, GSH provides neuroprotection, and Edward Lock discussed the selective toxicity of 2-chloropropionic acid (CPA) to the cerebellum and how its modification by modulating brain thiol status provides an example of GSH acting in neuroprotection. The sensitivity of the cerebellum to CPA may also be linked to the ability of this compound to activate a sub-type of the NMDA receptor. Thus, GSH and cysteine alone, or perhaps as conjugates with xenobiotics, may play a role in excitotoxicity via NMDA receptor activation. In contrast, certain chemicals may be converted to neurotoxicants following conjugation with GSH, and Arthur Cooper described how the pyridoxal 5'-phosphate-dependent, cysteine conjugate beta-lyases might predispose the brain to chemical injury in a GSH-dependent manner. The theme of GSH as a potential mediator of chemical-induced neurotoxicity was extended by Terrence Monks, who presented evidence for a role for GSH conjugation in (+/-)-3,4- methylenedioxyamphetamine-mediated serotonergic neurotoxicity.

Effects of S-ethyl hexahydro-1H-azepine-1-carbothioate (molinate) on di-n-butyl dichlorovinyl phosphate (DBDCVP) neuropathy.

Certain esterase inhibitors protect from organophosphate-induced delayed polyneuropathy (OPIDP) when given before a neuropathic organophosphate by inhibiting neuropathy target esterase (NTE). In contrast, they can exaggerate OPIDP when given afterwards and this effect (promotion) is associated with inhibition of another esterase (M200). In vitro sensitivities of hen, rat, and human NTE and M200 to the active metabolites of molinate, sulfone, and sulfoxide, were similar. NTE and M200 were irreversibly inhibited (> 78%) in brain and peripheral nerve of hens and rats given molinate (100-180 mg/kg, sc). No clinical or morphological signs of neuropathy developed in these animals. Hens and rats were protected from di-n-butyl dichlorovinyl phosphate neuropathy (DBDCVP, 1 and 5 mg/kg, sc, respectively) by molinate (180 or 100 mg/kg, sc, 24 h earlier, respectively) whereas 45 mg/kg, sc molinate causing about 34% NTE inhibition offered partial protection to hens. Hens treated with DBDCVP (0.4 mg/kg, sc) developed a mild OPIDP; molinate (180 mg/kg, 24 h later) increased the severity of clinical effects and of histopathology in spinal cord and in peripheral nerves. Lower doses of molinate (45 mg/kg, sc), causing about 47% M200 inhibition, did not promote OPIDP whereas the effect of 90 mg/kg, sc (corresponding to about 50-60% inhibition) was mild and not statistically significant. OPIDP induced by DBDCVP (5 mg/kg, sc) in rats was promoted by molinate (100 mg/kg, sc). In conclusion, protection from DBDCVP neuropathy by molinate is correlated with inhibition of NTE whereas promotion of DBDCVP neuropathy is associated with > 50% M200 inhibition.

Alpha 2U-globulin: measurement in rat kidney and relationship to hyaline droplets.

The concentration of renal alpha 2U-globulin increased in a dose-dependent manner in adult male but not female rats which received a single dose of 2,2,4-trimethylpentane (TMP). After administration of a single dose of 12 mmol TMP/kg to adult male rats, the renal concentration of alpha 2U-globulin reached a peak at 48 hours and returned to near background level after 7 days. These changes in renal alpha 2U-globulin concentration were closely paralleled by changes in renal hyaline droplet formation. Renal alpha 2U-globulin and hyaline droplets were absent in normal pre-puberty male rats, and neither could be stimulated by a single dose of TMP. alpha 2U-Globulin was localised in the renal cortex of adult male rats, in particular the S2 segment of the proximal tubule. A greater staining intensity due to alpha 2U-globulin was seen in the S2 and adjacent segments after a single dose of TMP. A strong association is suggested between the presence of renal hyaline droplets and the occurrence of alpha 2U-globulin.

An in vitro model of renal proximal tubule cell regeneration.

The ability of renal cells to regenerate is critical for the recovery of renal function following injury. Research on the recovery of renal function has been limited by the lack of in vitro models of renal repair. The goal of this study was to develop an in vitro model of renal proximal tubule cell (RPTC) injury and regeneration using primary cultures of rabbit RPTC. Renal proximal tubules were isolated and cultured in hormonally defined DME/F-12 medium at 37 degrees C under 95% air/5% CO2. RPTC were grown to confluency, made quiescent by the removal of insulin and hydrocortisone from the medium for 24-48 hr, and treated with the nephrotoxicant, 1,2-dichlorovinyl-L-cysteine (DCVC). DCVC (100 microM for 2 hr, n = 3-6) resulted in cell injury and the release of nonviable cells from the plate at 24 hr (55% +/- 6% confluency, mean +/- SEM) and 48 hr (37% +/- 7% confluency). Cell monolayers began to regenerate 96 hr after exposure (57% +/- 9% confluency) and continued to regenerate reaching 76% +/- 8% and 84% +/- 1% confluency by 6 and 8 days postexposure. Control cells maintained confluency throughout the experiment. Thus, an in vitro primary cell culture model has been developed in which the cell monolayer regenerates after nephrotoxicant-induced injury. This model may be useful in the study of mechanisms of renal cell injury and repair.

The accumulation and metabolism of 3-trifluoromethylpyridine by rat olfactory and hepatic tissues.

The accumulation of [methyl-14C]3-trifluoromethylpyridine (14C-3-FMP) by rat olfactory and hepatic tissue in vivo and in vitro has been investigated. 14C-3-FMP accumulates rapidly and selectively in both tissues in vivo, with an appreciable proportion of this activity being associated with the protein macromolecular fractions. Similar results were seen when isolated tissues were incubated in vitro in the presence of 14C-3-FMP. Studies with a range of metabolic inhibitors demonstrated that accumulation into olfactory tissue in vitro was virtually abolished by metyrapone and SKF-525A, indicating a key role of cytochrome P-450 mediated metabolism in this process. This was substantiated further by the in vivo inhibition of accumulation by metyrapone. Studies on the in vitro metabolism of 14C-3-FMP by isolated rat olfactory tissue demonstrated the major metabolite to be 3-trifluoromethylpyridine-N-oxide (3-FMP N-oxide) which is known to cause olfactory and hepatic toxicity in the rat. Metyrapone, while inhibiting accumulation of radioactivity derived from both 14C-3-FMP and 14C-3-FMP N-oxide in this tissue in vitro, only inhibited the synthesis of this metabolite by approximately 60%, indicating that several metabolic stages are involved in the metabolism and accumulation of 14C-3-FMP.

D-serine-induced nephrotoxicity: possible interaction with tyrosine metabolism.

D-serine selectively damages renal proximal tubule cells in rats by a mechanism that is not fully understood. Recent proteomic analysis identified that D-serine elevated plasma fumarylacetoacetate hydrolase (FAH). FAH is involved in tyrosine catabolism; hence, this pathway may be involved in mediating the toxicity. This work examines whether 2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione (NTBC), a potent inhibitor of the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD) located upstream of FAH, modulates D-serine-induced nephrotoxicity. Rats were pretreated with NTBC (0.5 mg/kg p.o.) or corn oil and then 30 min later given either D-serine (250 mg/kg i.p.) or water. Urine was collected every 12 h until termination (48 h) and analysed by 1H NMR spectroscopy and principal component analysis (PCA). Markers of proximal tubule injury were evident in urine following treatment with D-serine and NTBC + D-serine. PCA could not distinguish between these urine samples suggesting that NTBC does not effect the development of nephrotoxicity. Clinical chemistry analysis of urine and terminal plasma samples and histopathological examination of the kidneys confirmed this. NTBC alone caused a marked increase in the excretion of 4-hydroxyphenylpyruvate (HPPA) and 4-hydroxyphenyllactate (HPLA); however, HPPA and HPLA excretion was minimal following NTBC + D-serine. Instead marked tyrosinuria was observed suggesting that D-serine-induced renal damage markedly affects the handling of increased levels of HPPA and HPLA resulting from the inhibition of HPPD.

Investigations of the pathways of toxicity of methyl iodide in the rat nasal cavity.

The monohalomethane methyl iodide (MeI) is a site specific toxin within the nasal cavity of the rat, selectively damaging the olfactory epithelium (OE) whilst respiratory epithelium (RE) is spared. The aim of this study was to investigate the rates and routes of metabolism of MeI within the nasal cavity, in order to understand the reasons for the observed site-selectivity. Cytosolic glutathione S-transferases (GSTs) of both OE and RE catalysed the conjugation of MeI with glutathione (GSH), but rates were 4-fold higher in OE than RE. The product of this reaction was confirmed as S-methyl GSH. In both OE and liver the GST catalysing the conjugation of MeI was shown to belong to the theta class. No cytochrome P450-dependent oxidation of MeI to formaldehyde could be detected in incubations containing hepatic or olfactory microsomes. Intact nasal turbinates were incubated with [14C]-MeI, and a dose- and time-dependent covalent binding of MeI to olfactory protein was demonstrated. The rates of protein methylation were found to be similar in OE and RE. Thus the only parameter that correlates with the site-selectivity of the observed lesion is the rate of conjugation of MeI with GSH. Whether toxicity is due to production of a reactive metabolite or GSH depletion per se, remains to be elucidated.

Effects of 2-[1-(ethoxyimino)propyl]-3-hydroxy-5-(2,4,6-trimethylphenyl) cyclohex-2-enone on hepatic haem biosynthesis: species differences in hepatic porphyria.

2-[1-(Ethoxyimino)propyl]-3-hydroxy-5-(2,4,6-trimethylphenyl) cyclohex-2-enone (ETC) is a novel alkyl ketone herbicide. Continuous administration of ETC to mice for 28 days resulted in marked liver enlargement and severe intrahepatic cholestasis. These effects have been shown to result directly from a rapid and marked accumulation of porphyrin in the liver. The porphyrin which accumulates in the liver has been identified as protoporphyrin IX and dose response and time course studies confirm prior inhibition of mitochondrial ferrochelatase as the causal lesion. ETC was a very potent porphyrinogenic compound in mice, with a no-effect level for a single oral dose of 1 mg/kg. Rats and hamsters were insensitive to this type of hepatotoxicity following single oral doses of up to 750 mg/kg or following repeated, and indeed prolonged administration. The sensitivity of different species to ETC-induced porphyria correlated with the effect of ETC on hepatic ferrochelatase activity. The inhibition of ferrochelatase activity and the hepatic porphyria in mice were both found to be readily reversible upon withdrawal of ETC.

Alpha 2U-globulin: measurement in rat kidney following administration of 2,2,4-trimethylpentane.

Hyaline droplet formation was stimulated markedly in the kidneys of post-puberty male rats 24-48 h after a single oral dose of 12/24 mmol/kg 2,2,4-trimethylpentane [TMP]. Renal hyaline droplet formation could not be detected in female rats or in pre-puberty male rats following similar doses of TMP. A dose-dependent increase in the renal concentration of the androgen-dependent low molecular weight protein, alpha 2U-globulin was observed in post-puberty male rats 24 h after a single oral dose of TMP, over the range 0.3-12.0 mmol/kg. After administration of a single dose of 12 mmol/kg TMP to male rats, the renal concentration of alpha 2U-globulin rose steadily up to a peak after 48 h and then returned slowly to near normal after 7 days. Renal alpha 2U-globulin could not be detected in female rats and in pre-puberty male rats. An immunocytochemical assay was developed to examine the distribution of alpha 2U-globulin within the kidney. alpha 2U-Globulin was localised primarily in the S2 segment of renal proximal tubules in untreated male rats. Rats which received a single dose of 12 mmol TMP/kg showed not only a greater staining intensity, due to the presence of a higher concentration of alpha 2U-globulin, but also staining in adjacent segments of the renal cortex. Several urinary biochemical indicators of nephrotoxicity were measured daily in male rats for up to 72 h following a single dose of 12 mmol TMP/kg. Renal proximal tubular function was unimpaired by TMP treatment. On the basis of studies in untreated and TMP-treated rats, a strong association has been found between the presence of renal hyaline droplets and the occurrence of renal alpha 2U-globulin. The findings in the present study provide an explanation for the occurrence of renal hyaline droplets only in adult male rats, but do not, as yet, establish the toxicological significance of increases in renal hyaline droplet formation.