Reproducibility and robustness of metabolome analysis in rat plasma of 28-day repeated dose toxicity studies.

BASF has developed a rat plasma metabolomics database (MetaMap®Tox) containing the metabolome of more than 500 chemicals, agrochemicals and drugs, for which the toxicity is well known, derived from 28-day repeated dose toxicity studies in rats. The quality/reproducibility of data was assessed by comparing the metabolome of 16 reference compounds tested at least twice under identical experimental conditions at three time points (day 7, day 14 and day 28). Statistical correlation analysis showed that the repeated treatment induced very similar changes to the metabolome. For all repetitions the modes of action of the compounds were always correctly identified. Moreover, when compared against the metabolome of all compounds available in the MetaMap®Tox database, the repetitions showed in most cases the highest degree of overall similarity with the metabolome of the original study. In addition, we also evaluated the robustness of our metabolomics technique, displayed by constancy of variability in control groups over time. Based on these results, it can be concluded, that metabolomics can reproducibly be applied during toxicological in vivo testing in rats under the conditions applied here.

Increased toxicity when fibrates and statins are administered in combination--a metabolomics approach with rats.

Combination therapies with fibrates and statins are used to treat cardiovascular diseases, because of their synergistic effect on lowering plasma lipids. However, fatal side-effects like rhabdomyolysis followed by acute renal necrosis sometimes occur. To elucidate biochemical changes resulting from the interaction of fibrates and statins, doses of 100 mg/kg fenofibrate, 50mg/kg clofibrate, 70 mg/kg atorvastatin and 200 mg/kg pravastatin as well as combinations thereof were administered to Crl:Wi(Han) rats for 4 weeks. Plasma metabolome profile was measured on study days 7, 14 and 28. Upon study termination, clinical pathology parameters were measured. In a separate experiment plasmakinetic data were measured in male rats after 1 week of drug administration in monotherapy as well as in combinations. Lowering of blood lipid levels as well as toxicological effects, like liver cell degradation (statins) and anemia (fibrates) and distinct blood metabolite level alterations were observed in monotherapy. When fibrates and statins were co-administered metabolite profile interactions were generally underadditive or at the utmost additive according to the linear mixed effect model. However, more metabolite levels were significantly altered during combination therapy. New effects on the antioxidant status and the cardiovascular system were found which may be related to a development of rhabdomyolysis. Accumulation of drugs during the combination therapy was not observed.

In vitro metabolism, glutathione conjugation, and CYP isoform specificity of epoxidation of 4-vinylphenol.

4-Vinylphenol (4VP) has been identified as a minor urinary metabolite of styrene in rat and human volunteers. This compound has been shown to be more hepatotoxic and pneumotoxic than both styrene and styrene oxide at lower doses in rats and mice. To explore the possible toxicity mechanism of 4VP, the current study was conducted to investigate the metabolism of 4VP, the glutathione (GSH) conjugation of the metabolites of 4VP and its cytochrome P(450) (CYP) specificity in epoxidation in different microsomes in vitro. Incubations of 4VP with mouse lung microsomes afforded two major metabolites which were identified as 4-(2-oxiranyl)-phenol of 4VP (4VPO) and 4VP catechol. 4VPO was found to react with GSH to form GSH conjugate and 4VP catechol was found to further be metabolized to electrophilic species which react with GSH to form the corresponding 4VP catechol GSH conjugates. Relative formation rates for those GSH conjugates and the regioisomer formation of 4VPO-GSH conjugates with both inhibitors of CYP 2F2 and CYP 2E1 in microsomal incubation condition were also investigated. This present study provides better insight on the lung toxicity seen with 4VP, the toxic metabolite of commercial styrene.

Dermal uptake and excretion of 14C-toluene diisocyante (TDI) and 14C-methylene diphenyl diisocyanate (MDI) in male rats. Clinical signs and histopathology following dermal exposure of male rats to TDI.

Polyurethanes (PU) are polymers made with diisocyanates such as MDI (4,4'-methylene diphenyl diisocyanate) and TDI (2,4-toluene diisocyanate and 2,6-toluene diisocyanate). Investigations have been undertaken with MDI and TDI to assess dermal uptake and resulting systemic exposure. Absorption, distribution and excretion of MDI was studied in rats using a single dermal administration of (14)C-MDI dissolved in acetone at nominal 165 mg/kg body weight and 15 mg/kg bw (4.0 and 0.4 mg/cm(2)) and intradermal injection of (14)C-MDI dissolved in corn oil at nominal 1.4 mg/kg bw. Dermal absorption of (14)C-MDI (at both doses) was low; at or below 1% of the applied dose. Considerable amounts of the applied radioactivity were found at the application site which could not be washed off. By intradermal administration of (14)C-MDI approximately 66% of applied radioactivity remained at the application site with approximately 26% recovered in excreta, cage wash, tissues and carcass. The absorption, distribution and excretion of 2,4-TDI was studied in rats following a single dermal administration of radiolabelled (14)C-2,4-TDI at nominal 350 mg/kg body weight (12 mg/cm(2)). Dermal absorption of (14)C-2,4-TDI was at or below 1% of the applied dose. Considerable amounts of the applied radioactivity were found at the application site which could not be washed off. In summary the results show that dermal uptake of MDI and TDI is very low. Due to the chemical reactivity of isocyanates it can be expected that small amounts which might be absorbed will react with tissue constituents directly at the exposed skin area, or will be converted to adducts with biomacromolecules or to biologically inactive oligoureas. Overall it is concluded that, following dermal exposure to MDI and TDI, systemic exposures and resulting toxicity, other than the known sensitization, can be expected to be very low. In addition studies were performed with dermal application of unlabelled 2,4 and 2,6 TDI to check the availability and fate of this chemical on rat skin surface and to assess possible tissue damage. These experiments showed that unchanged test material can be detected on rat skin for up to 8h if not washed off. Dermal treatment with 2,4 or 2,6 TDI was associated with irritation with increased severity over a 48 h period after washing with a decontaminant solution.

The individual and combined metabolite profiles (metabolomics) of dibutylphthalate and di(2-ethylhexyl)phthalate following a 28-day dietary exposure in rats.

Metabolite profiles (metabolomics) of plasma samples of Wistar rats dosed with di(2-ethylhexyl)phthalate (DEHP - 3000ppm) and dibutylphthalate (DBP - 150, 1000 and 7000ppm) were individually determined in 28 days dietary studies. In addition, profiles of combined exposure to 3000ppm DEHP and either 150, 1000 or 7000ppm DBP were determined. High dose levels induced more profound metabolite changes in males than in females for both compounds. At 150ppm DBP (NOEL for toxicity) there were very few (

Influence of strain and sex on the metabolic profile of rats in repeated dose toxicological studies.

The impact of the strain on the metabolite profile of plasma samples in rats dosed with 2500 ppm 2-methyl-4-chlorophenoxyacetic acid (MCPA acid) or 45 mg/kg bw/day 4-chloro-3-nitroaniline (4C3N) for 4 weeks was evaluated. Four different strains were used: two Wistar strains (Crl:WI(Han), Han:RCC:WIST(SPF)), one Sprague-Dawley (Crl:CD) and one Fisher strain (F-344/Crl). The metabolite profiles in the plasma were measured by LC-MS and GC-MS. The profound changes of the metabolite values induced by the MCPA acid treatment outweighed slight deviations caused by physiological variations between the different rat strains. The metabolome changes of the MCPA acid in all strains could be related to toxicological "mode of action" patterns (peroxisome proliferator, renal organic anionic transporter inhibition) with Crl:WI(Han) rats as reference strain. 4C3N administration led to extravascular hemolytic anemia with a small number of metabolome changes, which were strain dependent. The metabolome pattern associated with "hemolytic anemia" established with the reference strain (Crl:Wi(Han)) was not sufficiently similar in other strains. Thus, comparable metabolome profiles were obtained in different rat strains for a compound inducing profound metabolite changes. For a compound with a weak profile the results were more variable and appeared to be strain dependent.

Generation and characterization of test atmospheres with nanomaterials.

To ensure the product safety of nanomaterials, BASF has initiated an extensive program to study the potential inhalation toxicity of nanosize particles. As preparation work for upcoming inhalation studies, the following manufactured nanomaterials have been evaluated for their behavior in an exposure system designed for inhalation toxicity studies: titanium dioxide, carbon black, Aerosil R104, Aerosil R106, aluminum oxide, copper(II) oxide, amorphous silicon dioxide, zinc oxide, and zirconium(IV) oxide. As the physicochemical properties and the complex nature of ultrafine aerosols may substantially influence the toxic potential, the particle size, specific surface area, zeta potential, and morphology of each of the materials were determined. Aerosols of each material were generated using a dry powder aerosol generator and by nebulization of particle suspensions. The mass concentration of the particles in the inhalation atmosphere was determined gravimetrically and the particle size was determined using a cascade impactor, an optical particle counter, and a scanning mobility particle sizer. The dispersion techniques used generated fine aerosols with particle size distributions in the respiratory range. However, as a result of the significant agglomeration of nanoparticles in the test materials evaluated, no more than a few mass percent of the materials were present as single nanoparticles (i.e., < 100 nm). Considering the number, a greater percentage of nanoparticles was present. Based on the obtained results and experience with the equipment, a technical setup for inhalation studies with nanomaterials is proposed. Furthermore, a stepwise testing approach is recommended that also could reduce the number of animals used in testing.

The use of metabolomics for the discovery of new biomarkers of effect.

Will metabolomics have a greater chance of success in toxicology and biomarker assessment than genomics and proteomics? Metabolomics has the advantage that (1) it analyses the last step in a series of changes following a toxic insult, (2) many of the metabolites have a known function and (3) changes are detectable in blood. If the analysis of a great number of individual organs can be replaced by one matrix then this will provide significant advantages (less invasive method, no need to kill animals, time course analysis possible). We have chosen to perform the analysis of blood metabolites in such a way as to minimize the risk of artifacts and to have a high number of known metabolites. We have also reduced the amount of variation in the biological system as well as during analysis. In a series of proof of concept studies it could be demonstrated that (1) the metabolome of control animals was stable of a period of nearly 1 year, with a remarkable differentiation between males and females, (2) a dose response relationship in metabolome changes was induced by phenobarbital and that (3) different modes of action could be distinguished by blood metabolome analysis. To investigate the potential of metabolomics to find biomarkers or specific patterns of change we have analyzed the blood metabolome of rats treated with HPPD inhibitors, a novel class of herbicides. The results demonstrated that a single metabolite, tyrosine, can be used as a biomarker. In addition to tyrosine we also found a specific pattern of change that involved nine metabolites. Though the extent of change was less than for tyrosine the consistent change of these metabolites is diagnostic for this (toxicological) mode of action.

Determination of pulmonary irritant threshold concentrations of hexamethylene-1,6-diisocyanate (HDI) prepolymers by bronchoalveolar lavage in acute rat inhalation studies according to TRGS 430.

Pulmonary irritant threshold concentrations of two hexamethylene-1,6-diisocyanate (HDI)-based prepolymers (I: polymeric emulsfier modified and II: oligomeric allophanate modified) were determined in acute inhalation studies according to TRGS 430 (Dangerous Substances Technical Rule, isocyanates, Germany), based on benchmark extrapolation of bronchoalveolar lavage fluid (BALF) total protein. It was also investigated if the method is robust enough to be transferred to an independent laboratory. Five male Wistar rats per group were exposed nose-only to the test substances as liquid aerosols to concentrations of 0, 0.5, 3, 15 mg/m(3) for both test substances with an additional test group at 50 mg/m(3) for test substance I. The duration of the exposure was 6h, followed by serial sacrifices 1 day, 3 days and 7 days post exposure. BALF was analyzed for biochemical and cytological markers indicative for injury of the bronchoalveolar region. The exposure of rats to test substance I and II caused dose depended lung irritation with BALF total protein concentration being the most sensitive indicator of pulmonary effects. The extrapolated no observed adverse effect level of test substance I was 1.1 mg/m(3) and that of test substance II 2.3 mg/m(3). The acute pulmonary irritant threshold concentrations were found to be similar to those reported by [Pauluhn, J., 2004. Pulmonary irritant potency of polyisocyanate aerosols in rats: comparative assessment of irritant threshold concentrations by bronchoalveolar lavage. J. Appl. Toxicol. 24, 231-247] for HDI-homopolymers and other HDI-based polyisocyanates, and were at least 30 times higher than the MAK (occupational exposure limit) value for the HDI monomer (0.035 mg/m(3)). Thus the EBW (exposure assessment value) for these two HDI-based prepolymers can be established at 10x MAK, i.e. at 0.35 mg/m(3).

The in vitro absorption of microfine zinc oxide and titanium dioxide through porcine skin.

Microfine metallic oxides such as titanium dioxide or zinc oxide have been found to be highly protective against harmful UV rays. Because their long-term use could potentially lead to health effects if significant amounts of these microfine metallic oxides would be absorbed through the skin, the in vitro absorption of microfine zinc oxide and titanium oxide in cosmetic formulations through porcine skin was investigated. In the experiments with a microfine zinc oxide formulation, the mean total recoveries of Zn were in the range from 102% to 107% of the total Zn applied. Virtually the total amount of applied Zn was recovered in the first five tape strips. The amounts of Zn found in the skin membrane and the receptor fluid were comparable in untreated, vehicle treated or test substance treated skin preparations. The absorption-time plots from diffusion cells treated with the vehicle did not differ from those treated with the ZnO containing formulation. In the experiments with microfine titanium dioxide formulations T-Lite SF-S and T-Lite SF, mean total recoveries of Ti ranged from 98% to 100% and 86% to 93% of the total Ti applied, respectively. Virtually the total amount of applied Ti could be removed from the skin surface by washing. The amounts of titanium found in the tape strips and skin preparations were in the order of the analytical determination limit. No Ti was found in the receptor fluid at any sampling time. The results show that neither zinc or titanium ions nor microfine zinc oxide or titanium dioxide particles were able to penetrate porcine stratum corneum. Therefore, from the absence of internal exposure we conclude that their use in sunscreens does not pose a health risk.

Absorption, distribution, metabolism and excretion of an inhalation dose of [14C] 4,4'-methylenediphenyl diisocyanate in the male rat.

The received dose, tissue distribution, metabolism, routes and rates of excretion of [(14)C]-4, 4(')-methylenediphenyl diisocyanate (MDI) were investigated in the male rat following a 6-h inhalation exposure to [(14)C]-MDI at a target concentration of 2 mg m(-3). The mean dose received was equivalent to 0.078 mg MDI per animal, of this between 25 and 32% of radiolabelled material was available systemically. Radioactivity was distributed to all tissues examined with the highest proportions present in the respiratory and gastrointestinal tracts, suggesting that both oral ingestion and pulmonary absorption contributed to the systemic dose of [(14)C]-MDI derived material, with the oral ingestion and the majority of the internal dose resulting from ingestion of radiolabelled material by grooming the pelt after exposure. Radioactivity was excreted mainly via faeces (about 80% of the received dose). Excretion in bile and urine each accounted for less than 15% of the dose. MDI was extensively metabolized after uptake, with two routes of transformation evident; the proposed spontaneous formation of mixed molecular weight polyureas and the enzyme catalysed metabolism of systemically available MDI or MDI derivatives to give N-acetylated and N-acetylated hydroxylated products. No free MDA was detected in any of the biomatrices (urine, faeces, bile) investigated.

A comparison between in vitro rat and human and in vivo rat skin absorption studies.

In vitro skin penetration rates in rat and man were compared to those obtained in vivo in rats. Saturation of absorption was frequently observed at higher exposure levels in in vitro and in vivo. Lipophilic compounds showed the highest penetration rates through rat skin in vitro. In all cases in vitro dermal penetration through rat skin was higher than in vivo. Thus, the in vitro study may serve as a first tier test. The in vivo data suggest an inverse relationship between molecular weight and the rate of dermal absorption for lipophilic as well as hydrophilic compounds. Rat skin was more permeable to all tested substances than human skin (mean difference 10.9-fold). Thus, the systemic exposure of humans may be significantly overestimated if risk assessment is based only on the results of an in vivo rat study, because human skin is less permeable than rat skin. It would appear, therefore, that an estimate of actual dermal penetration through human skin should be based on the combined use of in vivo and in vitro data, using the following equation: % Human dermal penetration = (% rat in vivo dermal penetration) x (in vitro rate dermal penetration human / in vitro rate dermal penetration rat).

The effects of styrene on lung cells in female mice and rats.

Styrene has been shown to cause an increase in the incidence of lung tumors in CD-1 mice following chronic exposure at 40 and 160 ppm, whereas no treatment-related increase in tumors in any organ was seen in rats chronically exposed to up to 1000 ppm styrene. So far most of the mechanistic studies have been performed with male animals. The aim of the present study was to further elucidate the target cell population in mouse lungs exposed to styrene, and to investigate possible differential in vivo effects (e.g., glutathione depletion, increased lipid peroxidation, and oxidative DNA damage). Groups of female CD-1 mice were exposed to styrene at concentrations of 0, 172 or 688 mg/m3 (0, 40 or 160 ppm) for 6 h per day on 1 day, 5 consecutive days or for 20 days during a 4 week period. Groups of female Crl:CD rats were exposed to styrene at concentrations of 0, 688 or 2150 mg/m3 (0, 160 or 500 ppm) for a single 6 h period or for 6 h per day on 5 consecutive days. No signs of lung toxicity were observed in rats. The cytology of cells in lung lavage fluid provided no signs of an inflammatory response in either rats or mice. In mice, both exposure levels caused decreased CC16 protein concentrations in lung lavage fluid after 1 and 5 exposures and in mouse blood serum throughout the study, suggesting that styrene may cause destruction of Clara cells in mice. Degenerative lesions in mouse Clara cells (vacuolar cell degeneration, cell necrosis) were revealed by electronmicroscopy. After 5 and 20 exposures of mice at 160 ppm, cellular crowding, expressed as an irregular epithelial lining and indicative of a very early hyperplasia was noted. Although a depletion of glutathione was noted in mouse lung homogenates after 20 exposures, there was no evidence of oxidative stress as indicated by unchanged concentrations of 8-OH-deoxyguanosine. Malondialdehyde, an indicator of lipid peroxidation, was slightly increased in mice after 1 exposure at 160 ppm only.

The significance of in vitro rat skin absorption studies to human risk assessment.

The present paper reviews the comparative rates of skin penetration between rat and man for a total of 14 chemicals in in vitro absorption studies. The results showed that in vitro absorption assays are capable of demonstrating large differences in the rate of skin penetration. Saturation of absorption was also frequently observed at higher exposure levels. The highest absorption rates through rat and human epidermis were observed with compounds with a molecular weight of approximately 300, an aqueous solubility of approximately 1-6 mg/l, and a log10 (P(OCTANOL/WATER)) of approximately 3-4. When the absorption data for 3 compounds with a log10 (P(OCTANOL/WATER)) of 2.9-3.0 were compared, there appeared to be an inverse relationship between molecular weight/aqueous solubility and the rate of dermal absorption. Lipophilic compounds with low aqueous solubility (<4 mg/l) showed the highest penetration rates through rat skin, but this was not always the case for human skin. The human skin was invariably less permeable to all tested substances than rat skin, though no constant factor of difference could be identified. The factor of difference would not appear to be determined by molecular weight, lipophilicity, or aqueous solubility. The actual systemic exposure of humans may be significantly overestimated if risk assessment is based only on the results of an in vivo rat study. It would appear that dermal penetration through human skin should be based on the combined use of in vivo and in vitro data, using the following equation: %Human dermal penetration= [[% dermal penetration rat (in vivo)] x [rate dermal penetration human (in vitro)]] / [rate dermal penetration rat (in vitro)]

Absorption, distribution, metabolism and excretion of 4-chloro-2-methylphenoxyacetic acid (MCPA) in rats.

The metabolism of 14C-MCPA (4-chloro-2-methylphenoxyacetic acid) in male and female rats was compared to that of 14C- MCPA dimethylamine salt (MCPA.DMA) or 14C-MCPA ethylhexyl ester (MCPA-EHE) in adsorption, distribution, metabolism and excretion studies. Compounds were administered by the oral route. The studies demonstrated the bioequivalence of the various forms of MCPA, established the extent of metabolism and metabolite identity. Following single or multiple oral administration of 5 mg/kg 14C-MCPA quantitative recovery of radioactivity, predominantly in urine, was obtained within 168 h. Rats dosed at 100 mg/kg showed similar absorption kinetics but apparent saturation of urinary excretion led to a prolonged elimination phase. MCPA was not extensively metabolised but the oxidation product HMCPA (4-chloro-2-hydroxymethylphenoxyacetic acid) was found at low levels, together with the glycine conjugate. These metabolites were more prominent shortly after dosing, suggesting that MCPA is not retained in the liver and that these metabolites may be excreted faster than MCPA itself. MCPA.DMA and MCPA-EHE were very rapidly converted into MCPA and toxicokinetics and metabolism were indistinguishable from parent compound.

Effect of cytochrome P-450 inhibition on tetrahydrofuran-induced hepatocellular proliferation in female mice.

The studies presented were designed to investigate the effects of cytochrome P450 inhibition on tetrahydrofuran-induced hepatocellular proliferation in female B6C3F(1) mice. Groups of female B6C3F(1) mice were exposed to dynamic atmospheres containing tetrahydrofuran (THF) concentrations of 0, 5,400 or 15,000 mg/m(3) for 6 h per day, for 5 consecutive days. One-half of the animals in each THF exposure group were pretreated with the cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) at 100 mg/kg (i.p.) 1 h before the start of each THF exposure period. Treatment with THF at 15,000 mg/m(3) caused marked microsomal enzyme induction in the liver. The cytochrome P450 content was nearly doubled (+98%), pentoxyresorufin-O-depentylase (PROD) and ethoxyresorufin-O-deethylase (EROD) activities were increased by 600% and 160%, respectively. ABT pretreatment effectively blocked microsomal enzyme induction at 15,000 mg/m(3). THF exposure had no effect on the subcellular morphology of hepatocytes, whereas ABT-pretreatment caused centrilobular fatty change. THF at 15,000 mg/m(3) caused increased cell proliferation in zone 3 (central vein region) of the liver (according to Rappaport), as indicated by a significantly higher PCNA (Proliferating Cell Nuclear Antigen) labelling index, but there were no effects at 5,400 ppm. ABT pretreatment prior to THF exposure at 15,000 mg/m(3) caused an exacerbated proliferative response of mouse liver, significantly higher PCNA labelling indices being observed in zones 2 (midzonal region) and 3. The exacerbated proliferative response of mouse liver under conditions of inhibited THF metabolism suggests that the mitogenic effects are related to prevailing THF tissue concentrations and not to the generation of THF oxidative metabolite(s).

NTA and Fe(III)NTA: differential patterns of renal toxicity in subchronic studies.

Differential patterns in terms of nephropathology and 8-hydroxyguanine formation in the course of oral 28-day studies were observed with nitrilotriacetic acid (NTA) and FeNTA. FeNTA, but not NTA, caused enhanced 8-hydroxyguanine formation in kidney DNA after oral and intraperitoneal administration. Enhanced lipid peroxidation in the kidney homogenate was observed with FeNTA as well as with NTA. For NTA, the low dose (9 mg/kg per day) was without adverse effect. The kidney toxicity of oral FeNTA (50, 200, and 1000 mg/kg per day) was only mild, 50 mg/kg per day; however, it still led to an increased 8-hydroxyguanine content. The relevance of Iron(III) (Fe(III)) or Fe(III)NTA formation as a relevant mediator of NTA-related toxicity was excluded on the basis of these data. Also, a thermodynamic consideration presented here, supports the view that zinc (Zn), and not Fe, is likely to mediate the tubular cell cytotoxicity of NTA.

Investigations on cell proliferation and enzyme induction in male rat kidney and female mouse liver caused by tetrahydrofuran.

To elucidate possible mechanism(s) of carcinogenic action of tetrahydrofuran (THF) that had been demonstrated in previous inhalation studies, groups of male F344 rats and female B6C3F(1) mice were exposed to dynamic atmospheric concentrations of 0, 600, 1800, or 5400 mg/m(3) for 6 h per day, either for 5 consecutive days or for a period of 4 weeks (5 days per week). The reversibility of treatment-related changes was investigated in rats and mice exposed for 5 days and sacrificed 21 days after the last exposure. Female B6C3F(1) mice exposed to 5400 mg/m(3) showed significantly increased cytochrome P450 content, increased ethoxyresorufin-O-deethylase and pentoxyresorufin-O-depentylase activities, increased cell proliferation (5-bromo-2'-deoxyuridine-method) and an increased mitotic index in liver zones 2 (midzonal region) and 3 (central vein region). The changes were found to be reversible after a 3-week treatment-free period (cell proliferation examined, only). Male F344 rats showed dose-related alpha2u-globulin (alpha2u) accumulation in the renal cortex after 5 or 20 exposures, and there were no signs of reversal after a 3-week treatment-free period. After 20 exposures at 5400 mg/m(3), the alpha2u accumulation was found to be associated with increased cell proliferation in "hot spots" of the renal cortex and increased apoptosis. Increased cell proliferation was also detected after 20 exposures at 1800 mg/m(3). There were no effects at 600 mg/m(3). It is concluded that THF enhances tumor formation in male rat kidney and female mouse liver via induction of cell proliferation. These features present essential elements that should be taken into account for the carcinogenic risk assessment of THF.

Immunolocalization of iron regulatory protein expression in the murine central nervous system.

We examined the expression of the iron regulatory proteins 1 and 2 (IRP1 and IRP2) in the brains of adult (4-6 months) CBA/J mice. Anti-IRP1 immunoreactivity was localized to cell bodies, including putative neurons and oligodendrocytes. In contrast, anti-IRP2 staining was prevalent throughout the neuropil of regions of the brain consistent with the central autonomic network (CAN) and mossy fibers emanating from hippocampal dentate granule cells. Essentially no staining for IRP2 was observed in the cerebellum in contrast to strong IRP1 immunoreactivity in Purkinje cells. Notably, cells within one vestibular nucleus exhibited staining by both IRP1 and IRP2. Our results suggest distinct roles for IRP1 and IRP2 in the regulation of iron homeostasis in the mammalian nervous system where IRP1 may provide a maintenance function in contrast to IRP2 that could participate in modulating proper CAN functions, including cardiopulmonary, gustatory as well as fine motor control.

Regulation of mammalian iron homeostasis.

Iron homeostasis is regulated with respect to uptake, storage and utilization. Newer work is presented that defines proteins responsible for iron transport, sequestration and sensing, and that addresses their regulation at the cellular and organismal levels by ambient iron concentrations, demand for erythropoiesis, body iron burden, and redox stimuli.