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Archives of Toxicology (v.78, #4)

Comparative responses of three rat strains (DA/Han, Sprague-Dawley and Wistar) to treatment with environmental estrogens by P. Diel; S. Schmidt; G. Vollmer; P. Janning; A. Upmeier; H. Michna; H. M. Bolt; G. H. Degen (pp. 183-193).

The rat uterotrophic assay is a widely used screening test for the detection of estrogenic, endocrine-disrupting chemicals. Although much attention has been paid to identifying protocol variables and reproducibility between laboratories the question whether toxicodynamic and toxicokinetic variations of different strains may affect their sensitivity to estrogenic stimuli has been rarely addressed. We have compared the estrogenic activity of the environmental chemicals genistein (GEN), bisphenol A (BPA) and p-tert-octylphenol (OCT) in DA/Han (DA), Sprague-Dawley (SD) and Wistar (WIS) rats after repeated oral application. Rats were treated per os for 3 days with different doses of these weakly estrogenic compounds and the potent reference estrogen ethinylestradiol (EE). Then uterine wet weight, thickness of the uterine epithelium, uterine gene expression of clusterin (CLU), and thickness of the vaginal epithelium were examined as parameters for estrogenic potency of the test compounds in the three strains of rats. The uterotrophic response to treatment with BPA, OCT and GEN was similar in the three strains, and allowed us to rank them as GEN being more potent than OCT, and BPA being the weakest estrogen. This was confirmed by analysis of other biological endpoints, despite some differences in the magnitude of their response among strains and to distinct compounds. For instance, the uterus wet weight response to EE treatment indicated lower sensitivity of SD rats than that of DA and WIS rats, but this was not observed for responses of the uterine or vaginal epithelium. Moreover, blood concentrations were assessed at the time of killing and related to biological responses: plasma levels of total and unconjugated BPA and GEN depended upon the dose administered and varied to some extent within treatment groups and among the three rat strains. However, there was no good correlation in the three strains between individual compound concentrations analysed 24 h after the last dose and the uterotrophic wet weights. Summarising our results, we conclude that the sensitivity of various biological endpoints can differ slightly between strains of rats. On the other hand, our data demonstrate that the choice of the rat strain does not lead to pronounced differences in the evaluation of estrogenic activities of chemicals, especially when different biological endpoints are included in the analysis.

Keywords: Bisphenol A; Clusterin; DA/Han rat; Genistein; p-tert-Octylphenol; Sprague Dawley rat; Uterus; Vagina; Wistar rat

Effects of chronic exposure to cadmium on renal cytochrome P450-dependent monooxygenase system in rats by Andrzej Plewka; Danuta Plewka; Grażyna Nowaczyk; Małgorzata M. Brzóska; Marcin Kamiński; Janina Moniuszko-Jakoniuk (pp. 194-200).

The aim of this study was to evaluate the effects of chronic exposure to cadmium (Cd) on the renal cytochrome P450-dependent monooxygenase system. For this purpose, male Wistar rats were intoxicated with Cd administered in drinking water at a concentration of 5 or 50 mg Cd/l for 6, 12 and 24 weeks. Concentrations of cytochrome P450 and cytochrome b₅ as well as activities of NADPH-cytochrome P450 reductase and NADH-cytochrome b₅ reductase were determined in the kidney microsomal fraction. Protein content of CYP1A1, CYP2E1 and CYP3A1 cytochrome P450 isoforms was evaluated as well. In the rats exposed to 5 mg Cd/l, the concentration of cytochrome P450 decreased (by 41%) after 24 weeks of the experiment. The activity of NADPH-cytochrome P450 reductase decreased (by 24%) after 6 and 12 weeks, whereas after 24 weeks it remained unchanged, compared with the control group. Moreover, a decrease in the concentration of cytochrome b₅ (by 25, 15 and 26% at 6, 12 and 24 weeks, respectively) and the activity of its NADH reductase (by 26 and 31% at 6 and 24 weeks, respectively) was noted in these animals. At the exposure to 50 mg Cd/l, the concentrations of cytochrome P450 and cytochrome b₅ and the activities of their corresponding reductases were decreased at each time-point. Western blot analysis revealed that all isoforms of cytochrome P450 studied were affected by Cd and the effect was dependent on the level and the duration of exposure. The results of this study indicate that chronic exposure to Cd in a dose- and time-dependent manner affects the kidney cytochrome P450-dependent monooxygenase system by decreasing the concentrations of cytochrome P450 and cytochrome b₅ and inhibiting the activities of their corresponding reductases. The effect of Cd on the cytochrome P450 content is associated with its ability to stimulate or inhibit of various P450 isoforms. A very important finding of this study is that Cd affects the kidney cytochrome P450-dependent monooxygenase system at relatively low exposure and low kidney Cd accumulation (2.40±0.15 μg/g). As the experimental model used reflects human exposure to Cd, we conclude that Cd can affect the kidney cytochrome P450-dependent monooxygenase system in environmentally exposed humans. Previously we have reported disorders in the system in the liver of rats at the same levels of exposure as in this study. Thus, we hypothesize that the metabolism and detoxification of many substances, including xenobiotics, may be seriously affected in Cd-exposed subjects.

Keywords: Cadmium; Cytochrome P450; P450 isoforms; Cytochrome b₅ ; Kidney; Rats

Comparison of the sensitivity of different toxicological endpoints in Caco-2 cells after cadmium chloride treatment by Monica Boveri; Patricia Pazos; Alessandra Gennari; Juan Casado; Thomas Hartung; Pilar Prieto (pp. 201-206).

The human colorectal adenocarcinoma cell line Caco-2 is a widely used in vitro model of the intestinal barrier. Cadmium chloride (CdCl₂) is a highly toxic metal compound, ubiquitous in the biosphere, able to enter the food chain and to reach the intestinal epithelium, causing structural and functional damages. The aim of this work was to characterise cadmium toxicity in Caco-2 cells and, in particular, to compare the sensitivity of different endpoints revealing damage both on the epithelial barrier and at the cellular or molecular level. After 24-h exposure of the cells to CdCl₂, lactate dehydrogenase (LDH) leakage showed cadmium-induced cell toxicity, significant from 25 µM CdCl₂ and above, and analysis of different cell death pathways indicated the presence of necrosis after treatment with 50 µM CdCl₂. At the molecular level, we observed an increase in the protective protein heat shock protein 70 (HSP70), starting at 10 µM CdCl₂. At the barrier level, transepithelial electrical resistance (TEER) decreased while paracellular permeability (PCP) significantly increased after the treatment, showing an EC₅₀ of 6 and 16 µM CdCl₂, respectively, and indicating the loss of barrier integrity. In conclusion, our data reveal that CdCl₂ toxicity in Caco-2 cells can be detected at the barrier level at very low concentrations; also, HSP70 was shown to be a sensitive marker for detecting in vitro cadmium-induced toxicity.

Keywords: Caco-2; Cadmium; In vitro; Intestinal toxicity

Neurobehavioural test results and exposure to inorganic mercury: in search of dose-response relations by Monika Meyer-Baron; Michael Schaeper; Christoph van Thriel; Andreas Seeber (pp. 207-211).

The aim of the analysis was, in general, to find a way to summarise results of studies in search of exposure-response relationships and, in particular, to ask whether an exposure-response relationship can be ascertained for neurobehavioural studies on occupational mercury exposure. Eighteen studies dealing with human mercury exposure and examining 1,106 exposed and 1,105 control subjects were included in the analysis. Effect sizes were calculated for each of the single neuropsychological test results on cognitive and motor performance and were considered in relation to mean current concentrations of exposure. The total of effect sizes demonstrated a correlation to exposure in the range of r=0.50. Additional analyses showed that the influence of mercury on psychological functions was different and that results on motor performance compared with memory and attention revealed the greatest impairment in mercury-exposed workers. Implications for the reversibility of impairments could be discussed because three of the studies examined subjects whose exposure had ceased. Besides the usefulness of the approach, it became obvious that the available 18 studies are a small sample, even when non-dimensional effect-sizes are used; hence, not all psychological domains covered by tests could be analysed.

Keywords: Inorganic mercury; Neurotoxicity; Dose-response relationship; Risk assessment; Cognitive functions

Reactivation and aging kinetics of human acetylcholinesterase inhibited by organophosphonylcholines by F. Worek; H. Thiermann; L. Szinicz (pp. 212-217).

A great number of structurally different organophosphorus compounds (OPs) was synthesized in the past decades to be used as pesticides or chemical warfare agents. Methyl-fluorophosphonylcholines were found to be highly toxic OPs and the acetylcholinesterase (AChE) reactivator pralidoxime was shown to be unable to reactivate inhibited AChE. In the course of the development of more effective AChE reactivators, we have determined the reactivation rate constants of various oximes with human AChE inhibited by methylfluorophosphonylcholine (MFPCh), methylfluoro-β-phosphonylcholine (MFPβCh) and methylfluorophosphonylhomocholine (MFPhCh). In addition, we investigated the potential influence of aging phenomena on the oxime efficacy. Human AChE inhibited by MFPCh, MFPβCh or MFPhCh was extremely resistant towards reactivation by oximes. Nevertheless, the newer compounds, HLö 7 and HI 6, were substantially more potent reactivators than obidoxime and pralidoxime. The low oxime efficacy was not due to rapid aging since no decrease in reactivatability was found over 96 h at 37°C. Within this period a substantial spontaneous reactivation was observed, with MFPCh >MFPβCh >MFPhCh, which did not follow pseudo-first-order kinetics. In conclusion, the unexpected results, i.e., high resistance of inhibited AChE towards oxime reactivation and aging, and much lower resistance towards spontaneous reactivation, calls for further experiments at a molecular level for a better understanding of the interactions among AChE, its inhibitors and reactivators.

Keywords: Organophosphate; Acetylcholinesterase; Oximes; Reactivation kinetics; Aging

Low glutathione S-transferase dogs by Toshiyuki Watanabe; Tomomi Sugiura; Sunao Manabe; Wataru Takasaki; Yoshihiko Ohashi (pp. 218-225).

Liver and kidney glutathione S-transferase (GST) activities to 1,2-dichloro-4-nitrobenzene (DCNB) as a substrate (GST-D activities) were measured in 280 dogs from five different breeders, and significant individual differences in this activity were observed in both organs. Interestingly, 34 out of the 280 dogs (i.e. 12.1%) were those in which liver GST-D activities were less than 10 nmol/min per mg cytosolic protein, “low GST dogs”, and the other dogs were classified as “middle” and “high” GST dogs for which the liver GST-D activities were 10–80 and >80 nmol/min per mg protein, respectively, and occurred at similar percentages (41.4% for the middle GST dog and 46.4% for the high GST dog). Furthermore, the existence of the low GST dogs was not limited to one particular breeder. There was a good correlation (r=0.910) between the liver and kidney GST-D activities, showing low activity in not only the liver but also the kidney in the low GST dogs. Although liver GST activity to 1-chloro-2,4-dinitrobenzene as a substrate (GST-C activity), catalyzed by various GST isozymes in dogs, was significantly correlated with liver GST-D activity, GST-C activity showed more than 450 nmol/min per mg protein even in the low GST dogs. There was no significant difference in cytochrome P450 content, 7-ethoxycoumarin O-deethylase activity or UDP-glucuronosyltransferase activity to p-nitrophenol as a substrate between low GST dogs and the other dogs. Finally, remarkably high plasma concentrations of DCNB were observed in the low GST dogs after single doses of DCNB at 5 or 100 mg/kg. The individual differences in GST-D activity are probably attributable to the content and/or activity of the theta class GST isozyme Yd_fYd_f since it has been reported that glutathione conjugation of DCNB is specifically catalyzed by GSTYd_fYd_f in dogs. In conclusion, we identified a number of low GST dogs in which the GST-D activities were not observed either in vivo or in vitro. The feasibility of using a single low dose of DCNB to phenotype dogs based on GST-D activity was confirmed. It was also suggested that low GST dogs have high susceptibility, including unexpected toxicity or abnormal exposure, to chemicals metabolized by GSTYd_fYd_f.

Keywords: Glutathione S-transferase; Low GST dog; GSTYd_fYd_f ; Deficiency; High susceptibility

Changes in the structure and function of the kidney of rats chronically exposed to cadmium. II. Histoenzymatic studies by Małgorzata M. Brzóska; Marcin Kamiński; Mirosław Dziki; Janina Moniuszko-Jakoniuk (pp. 226-231).

Early effects of cadmium (Cd) on the structure and function of the kidney were studied in an experimental model using rats intoxicated with Cd at the levels of 5 and 50 mg Cd/l drinking water. The effect of Cd was evaluated histopathologically and biochemically. Damage to the cellular structures was assessed on the basis of histoenzymatic analyses of the activity and localization of indicator enzymes (succinate dehydrogenase, lactate dehydrogenase, glucose-6-phosphatase, Mg²⁺-dependent adenosine triphosphatase and acid phosphatase). The histochemical observations indicate that Cd causes damage to the organization and function of the nephron. Several structures, i.e. endoplasmic reticulum, mitochondrion, lysosome, cellular and intracellular membrane, as well as their biological functions, i.e. aerobic and anaerobic respiration, transport functions and biochemical processes taking place in the endoplasmic reticulum, were affected. The cytotoxic action of Cd occurs mainly in the tubules and partially also in the glomeruli. The results clearly indicate that Cd damages kidney structurally and functionally even at a relatively low level (5 mg/l) corresponding to human environmental exposure, and they confirm our previous hypothesis that the threshold for the kidney effects of Cd is less than 4.08±0.33 μg/g kidney wet weight and higher than 2.40±0.15 μg/g. The target for Cd action in the kidney is the tubules (proximal convoluted tubules and straight tubules), and disturbance in their function is the main toxic effect of Cd. Renal glomeruli are also injured, but only partially, whereas in other parts of the nephron the damage is slight. The results, together with observations reported in the first paper of the study, incline us to conclude that humans environmentally exposed to Cd are at risk of tubular damage.

Keywords: Cadmium; Kidney; Marker enzymes; Histochemistry; Rats

Alteration of pituitary hormone-immunoreactive cell populations in rat offspring after maternal dietary exposure to endocrine-active chemicals by Naoya Masutomi; Makoto Shibutani; Hironori Takagi; Chikako Uneyama; Kyoung-Youl Lee; Masao Hirose (pp. 232-240).

We previously performed dose–response studies of genistein, diisononyl phthalate, 4-nonylphenol, methoxychlor (MXC), and bisphenol A to examine the impact of maternal dietary exposure from gestational day 15 to postnatal day 10 on the development of rat reproductive system in later life. Among the chemicals MXC alone showed typical estrogenic effects only at the maternally toxic 1200 ppm. The present study was performed to examine the sensitivity of immunohistochemical analysis of pituitary cells of offspring similarly exposed to each chemical for detection of endocrine-disrupting effects. For this purpose, ratios of pituitary cells expressing luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL), were measured at 3 and 11 weeks of age. Ethinylestradiol (EE) at 0.5 ppm was used as a reference chemical. At week 3, decrease in the relative proportions of LH, FSH, and PRL cells in males and LH cells in females was evident with MXC at 1200 ppm. At week 11, increase was found for PRL cells from 240 ppm MXC, and FSH cells at 1200 ppm in females. On the other hand, EE increased the PRL cell percentage in females at week 3 but no effects were apparent at week 11. The other chemicals were without influence at either time point. The results suggest that the assessment of the pituitary cell populations might be a more sensitive approach to detect perinatal endocrine-disrupting effects than other methods. The difference in the pituitary effect between MXC and EE is discussed.

Keywords: Endocrine-acting chemicals; Perinatal exposure; Pituitary hormone; Rat; Immunohistochemistry; Genistein; Diisononyl phthalate; 4-Nonylphenol; Methoxychlor; Bisphenol A; Ethinylestradiol

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Archives of Toxicology (v.78, #4)