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Archives of Toxicology (v.72, #9)

Relationship between the development of hepato-renal toxicity and cadmium accumulation in rats given minimum to large amounts of cadmium chloride in the long-term: preliminary study by K. Mitsumori; M. Shibutani; S. Sato; H. Onodera; J. Nakagawa; Y. Hayashi; M. Ando (pp. 545-552).

We wished to clarify the relationship between the sensitivity to induce hepato-renal toxicity and the level of cadmium (Cd) in the organs of rats exposed to minimum to large amounts of cadmium chloride (CdCl₂). For this purpose, groups of female Sprague-Dawley (SD) rats, each consisting of 24 animals, were fed diet containing CdCl₂ at concentrations of 0, 8, 40, 200, and 600 ppm for 2, 4, and 8 months from 5 weeks of age. All surviving rats given 600 ppm Cd were killed at 4␣months because of deterioration of their general condition. Animals of this group showed anemia and decreased hematopoiesis in the bone marrow, in addition to reduction of cancellous bone in their femurs. Hepatotoxicity was observed after 2 months in the groups treated with 200 ppm. By 4 months, the rats in the 600 ppm group had developed periportal liver cell necrosis. Renal toxicity characterized by degeneration of proximal tubular epithelia was apparent in the groups treated with 200 ppm from 2 months, becoming more prominent in the high-dose rats at 4 months. Hepatic accumulation of Cd increased linearly with the duration of treatment. In contrast, the concentration of Cd in the renal cortex of rats treated with 600 ppm reached a plateau level of ∼250 μg/g within the first 2 months. The renal concentration of Cd in the 200 ppm group when renal toxic lesions were first detected at 2 months ranged from 104 to 244 μg/g. No renal lesions were observed in the 40 ppm group after 8 months, despite the presence of 91–183 μg/g of Cd in the kidneys. The results thus suggest that renal toxicity would not be induced by treatment with minimum amounts of CdCl₂ for periods longer than 8 months, although accumulation of Cd might gradually progress. A further 2-year feeding study of CdCl₂ and Cd-polluted rice is now in progress.

Keywords: Key words CdCl2; Chronic toxicity; Body burden; of Cd; Renal toxicity; Liver toxicity

The role of glutathione and cysteine conjugates in the nephrotoxicity of o-xylene in rats by G. Morel; P. Bonnet; B. Cossec; S. Morel; C. Cour; A. M. Lambert; M. B. Roure; M. T. Brondeau (pp. 553-558).

Moderate nephrotoxicity was induced in male and female rats exposed to o-xylene for 4 h at atmospheric concentrations of ∼3000 ppm. The xylene in␣vivo nephrotoxicity resulted in low enzyme leakage from the kidney into the urine. This low leakage was confirmed in 24-h urine by an increase in γ-glutamyltranspeptidase (γGT), N-acetyl-β-d-glucosaminidase (NAG) and alkaline phosphatase (ALP) activities. Compared to the control, both the 24-h urine output and the glucose excretion increased in male and female rats. These increases were probably a result of damage to the renal proximal tubules. The role of the metabolic pathway of glutathione in the emergence of the renal damage observed with o-xylene was investigated in rats. Recent studies indicate that the metabolic pathway of glutathione may be a bioactivation pathway, which is responsible for nephrotoxic effects with several drugs or chemicals. The renal toxicity of three synthesized o-xylene thio-conjugates was investigated in several groups␣of female rats. Administration of S-(o-methylbenzyl)glutathione (i.p., 1 mmol/kg), S-(o-methylbenzyl)cysteine (per os, 1 mmol/kg) or N-acetyl-S-(o-methylbenzyl)cysteine (i.p., 0.75 mmol/kg) to female rats did not induce renal toxicity, as monitored by urinary biochemical parameters (γGT, NAG, ALP, glucose). The data obtained suggest that the glutathione pathway would appear to be only detoxication, and probably does not contribute to the renal toxicity of o-xylene in female rats. Thus, either another metabolic pathway or other intermediate metabolites are probably involved in the nephrotoxic action of o-xylene.

Keywords: Key wordso-Xylene; Nephrotoxicity; Metabolism; Glutathione

Cyclosporin A induces apoptosis in rat hepatocytes in culture by Irene D. Román; Nieves Rodríguez-Henche; Jesús A. Fueyo; Jose A. Zueco; César Menor; Juan C. Prieto; Luis G. Guijarro (pp. 559-565).

Cyclosporin A (CsA) at concentrations up to 1 μM induced apoptosis in a dose-dependent manner in cultured rat hepatocytes for 48 h in the presence of insulin and epidermal growth factor (EGF). The effect of CsA was evidenced by the DNA fragmentation pattern constituted by fragments of multiples of 180–200 base pairs, which is a characteristic of programmed cell death. The metabolic activity did not change significantly in the presence of 0.1 μM CsA and diminished to 49% of control in the presence of 1 μM CsA. Changes in the metabolic activity were correlated with a decrease in both [methyl-³H]thymidine uptake and DNA content, which reflects a decrease in the cell number. The treatment of cells with CsA (1 μM) decreased the metabolic activity/DNA content ratio by 24% with respect to dimethyl sulphoxide (DMSO) control, which also suggests, under these conditions, that the necrosis achieved is at most only 24%. In addition, the changes observed (apoptotic process, arrest of the cell cycle and apparition of a necrotic process) were correlated with an increase in the high-affinity guanosine triphosphatase (GTPase) enzymes.

Keywords: Key words Proliferation; Epidermal growth factor (EGF); Insulin; Hepatocyte; Cyclosporin A

Different effects of inorganic and dimethylated arsenic compounds on cell morphology, cytoskeletal organization, and DNA synthesis in cultured Chinese hamster V79 cells by Takafumi Ochi; Fumie Nakajima; Nobutaka Fukumori (pp. 566-573).

Changes in cytoskeletal organization of cultured V79 cells exposed to arsenite and dimethylarsinic acid (DMAA), a methylated derivative of inorganic arsenics, and related changes, such as mitotic arrest and induction of multinucleated cells, were investigated in comparison with their effects on DNA synthesis. DMAA caused mitotic arrest and induction of multinucleated cells with a delay of 12 h relative to the mitotic arrest. By contrast, arsenite at equitoxic concentrations to DMAA was less effective than DMAA in causing mitotic arrest and in inducing multinucleated cells. Post-mitotic incubation of cells arrested in metaphase by 6 h incubation with 10 mM DMAA showed that the incidence of multinucleated cells increased conversely with a rapid decrease in metaphase cells. This suggests that metaphase-arrested cells can escape from metaphase, resulting in the appearance of multinucleated cells. The mitotic arrest caused by DMAA was accompanied by disruption of the microtubule network. By contrast, both arsenite and DMAA did not cause disorganization of actin stress fibers even when incubated at concentrations that caused a marked retardation of cell growth. Cells exposed to arsenite for 6 h showed marked inhibition of DNA synthesis, whereas inhibition by DMAA was not observed. When incubation was prolonged by 18 h, the arsenite-induced inhibition of DNA synthesis was mitigated. By contrast, inhibition of DNA synthesis by DMAA occurred in parallel with an increase in the population of mitotic cells. These results suggest that DMAA caused growth retardation and morphological changes via disruption of the microtubule network, and that arsenite-induced retardation of cell growth and inhibition of DNA synthesis were not attributable to the cytoskeletal changes.

Keywords: Key words Arsenic compounds; Microtubule network; Mitotic arrest; Multinucleated cells; DNA synthesis

Modulation of nonprotein sulphydryl compounds rhythm with buthionine sulphoximine: relationship with oxaliplatin toxicity in mice by Xiao-Mei Li; Gérard Metzger; Elisabeth Filipski; Guy Lemaigre; Francis Lévi (pp. 574-579).

The relationship between the rhythm in tissue nonprotein sulphydryl groups (NPSH) and that in 1,2-diamine (trans-I)-cyclohexane oxalatoplatinum (l-OHP) toxicity was investigated in a total of 266 male B₆D₂F1 mice, using buthionine sulphoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase. Mice were synchronized with an alternation of 12 h light (L) and 12 h darkness (D; LD 12:12), and circadian time was expressed in hours after light onset (HALO). NPSH was measured in liver, jejunum and bone marrow at 0, 8 and 16 HALO. Dosing l-OHP at these times achieved intermediate, high or low toxicity respectively. The physiological circadian rhythm in NPSH content was statistically significant in all tissues studied, with a maximum at the transition from D to L (0 HALO). BSO administration (450 mg/kg i.p., 4 h before sampling) induced a large depletion in liver and jejunum NPSH at their physiological peak (0 HALO), but exerted no significant effect at their trough (8 HALO). As a result, 24 h rhythm was suppressed in liver and jejunum, but remained similar to the physiological one in bone marrow. BSO enhanced l-OHP-induced mortality and jejunal toxicity, but exerted no significant effect upon bone marrow toxicity. Despite these differences, l-OHP remained least toxic at 16 HALO, near the middle of the dark span, which corresponds to maximum activity in the circadian rest/activity cycle. Our results show that mean NPSH levels in liver seem to account for the mean level of l-OHP toxicity, while jejunal NPSH rhythm plays an important role in the intestinal toxicity rhythm of this drug.

Keywords: Key words Circadian rhythm; Oxaliplatin; Sulphydryl compound; Toxicity

Inhibition, reactivation and aging kinetics of cyclohexylmethylphosphonofluoridate-inhibited human cholinesterases by F. Worek; P. Eyer; L. Szinicz (pp. 580-587).

Cyclohexylmethylphosphonofluoridate␣(cyclosarin) is a highly toxic organophosphate, which was shown to be rather resistant to conventional oxime therapy. To give more insight into the inhibition, reactivation and aging kinetics, human acetyl-(AChE) and butyrylcholinesterase (BChE) were inhibited by cyclosarin (k ₂ of 7.4 and 3.8 * 10⁸ M⁻¹ min⁻¹, respectively; pH 7.4, 37 °C) and reactivated with obidoxime, pralidoxime and three experimental oximes. The new oxime HLö 7 (1-[[[4-aminocarbonyl)-pyridinio]-methoxy]-methyl]-2,4-bis-[(hydroxyimino)methyl] pyridinium dimethanesulphonate) was shown to be superior to the other oximes. At oxime concentrations anticipated to be relevant in humans, obidoxime and pralidoxime were extremely weak reactivators of AChE. Aging velocity of BChE was almost fourfold higher compared to AChE (k_a of 0.32 h⁻¹ and 0.08 h⁻¹, respectively). A substantial spontaneous reactivation was observed with AChE. These results support previous in vivo findings that obidoxime and pralidoxime are insufficient antidotes in cyclosarin poisoning. By contrast, HLö 7 was shown to be an extremely potent reactivator of human AChE and BChE,␣which supports its position as a broad-spectrum oxime.

Keywords: Key words Cyclosarin; Acetylcholinesterase; Butyrylcholinesterase; Inhibition; Reactivation

Ethionine toxicity in vitro: the correlation of data from rat hepatocyte suspensions and monolayers with in vivo observations by Catherine J. Waterfield; Carl Westmoreland; Daniel S. Asker; Janice C. Murdock; Elisabeth George; John A. Timbrell (pp. 588-596).

The hepato-steatogenic compound ethionine has been used to investigate the correlations between in␣vivo and in vitro toxicity data. The aim was to find a suitable model of toxicity in hepatocyte suspensions or monolayers in vitro, which could predict the known toxicity of ethionine in vivo and which could be implemented in screening compounds of unknown toxicity. Thus a variety of markers of cytotoxicity, metabolic competence and liver-specific functions were investigated in rat hepatocyte suspensions and monolayers and compared with in vivo data in the rat. The following markers were measured in the appropriate system: (1) Neutral red uptake; 3-(4,5 dimethyl)thiazol-2-yl,-2,5-diphenyl tetrazolium bromide (MTT) reduction; lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) leakage (cytotoxicity). (2) ATP levels, protein synthesis and glutathione (GSH) levels (metabolic competence). (3) Urea and triglyceride synthesis and β-oxidation (liver specific functions). Ethionine (0–30 mM) did not affect the markers of direct cytotoxicity, except neutral red uptake, which was reduced by 18 and 30 mM ethionine after 20 h in culture. ATP and GSH depletion occurred in hepatocyte suspensions at the highest concentrations of ethionine (20 and 30 mM) after 1 h. In monolayers, GSH levels were reduced after 4 h, but not 20 h. Urea synthesis was increased in hepatocyte suspensions from 1 to 3 h by 10–30 mM ethionine and reduced after 20 h in cultured hepatocytes (18–30 mM). Protein synthesis was reduced and β-oxidation was increased in ethionine-treated hepatocyte suspensions. Unfortunately, there was no measurable effect on triglyceride accumulation within cells (the major biochemical change in␣vivo) in either system. Ethionine treated hepatocytes in suspension showed the same rate of triglyceride synthesis and transportation out of cells as control cells. Thus, hepatocyte suspensions were able to mimic the early biochemical effects of ethionine in vivo (ATP and GSH depletion, inhibition of protein synthesis) and some effects on urea synthesis, but monolayer cultures appeared to be less sensitive to the toxicity of ethionine. However, neither in vitro system was able to model the effects of ethionine on the accumulation of triglycerides in vivo.

Keywords: Key words In vivo/in vitro correlations; Ethionine; Hepatocytes

2,5-Hexanedione concentrations and morphological changes within the eye of albino rat by Birgitta Bäckström; Eiji Shibata; Per Nylén; V. Peter Collins (pp. 597-600).

Exposure to 2,5-hexanedione (2,5-HD), a major n-hexane metabolite, can cause loss of photoreceptor cells, particularly when combined with light energy. The aims of this study were to document the levels of 2,5-HD reached in relation to time in retina, aqueous humor, and serum of the Sprague-Dawley albino rat after: (1) a single oral administration of 2,5-HD (0.04 g/kg body wt.) by tube feeding; and (2) after subchronic oral administration of 2,5-HD. In addition, morphometric analysis of the retina was carried out to evaluate cell loss at the end of administration and after various periods of recovery. The 2,5-HD concentration in retinal tissue, aqueous humor, and serum reached a peak within 1 h after exposure to a single dose of 2,5-HD. Twenty four hours after the exposure, only a minor amount of 2,5-HD could be detected in the retina and aqueous humor. When 2,5-HD was administered subchronically (0.04 g/kg body wt. per day, for 35 days) no loss of photoreceptor cells was seen immediately after the end of exposure or at the end of a 4-week recovery period. Rats exposed to 0.60 g/kg body wt. per day for 11 days showed no significant loss of photoreceptor cells immediately after the end of exposure but there was a substantial loss of photoreceptor cells after 2 and 4 weeks of recovery. The results demonstrate that 2,5-HD reaches the aqueous humor and retina, and penetrates blood-aqueous humor/retina barriers after oral administration. Moreover, retinal degeneration seen in the animals may be directly caused by 2,5-HD and these changes are dose dependent.

Keywords: Key words 2; 5-Hexanedione ; Aqueous humor ; Photoreceptors ; Retina ; Serum

Urinary metabolites of sarin in a patient of the Matsumoto sarin incident by T. Nakajima; K. Sasaki; H. Ozawa; Y. Sekijima; H. Morita; Y. Fukushima; N. Yanagisawa (pp. 601-603).

Sarin metabolites were measured in urine from a patient with sarin poisoning. Two metabolites, methylphosphonic acid (MPA) and isopropylmethylphosphonic acid (iPMPA), were detected by gas chromatography after conversion to volatile derivatives with N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide in the urine from the victim collected on the first day of hospitalization. iPMPA was detected in the urine on the seventh day, but MPA could not be detected in the urine sample. MPA was narrowly detected in the urine collected on the third day. The concentration of iPMPA was estimated on the assumption that the sensitivity of phosphorus was the same as that of MPA. The total excretion of iPMPA and MPA in the urine was 2.1 mg and 0.45 mg, respectively. When all the sarin inhaled was excreted within a week as these two metabolites, the subject was considered to have been exposed to 2.79 mg (0.05 mg/kg) sarin at the incident. Thus, the measurement of sarin metabolites in urine is a useful tool for the biological monitoring of exposure to sarin.

Keywords: Key words Sarin; Methylphosphonic acid; Isopropylmethylphosphonic acid; Biological monitoring; Urinary metabolites

In vitro studies of human alcohol dehydrogenase inhibition in the process of methanol and ethylene glycol oxidation by Katarzyna Dawidek-Pietryka; Stanisław Szczepaniak; Jarosław Dudka; Marek Mazur (pp. 604-607).

The liver is the major organ responsible for methanol and ethylene glycol oxidation, and alcohol dehydrogenase (alcohol: NAD⁺ oxidoreductase, EC 1.1.1.1.) is the main enzyme involved. In the present study, alcohol dehydrogenase (ADH) activity was measured spectrophotometrically in vitro at physiological pH 7.4 and 37 °C using human enzyme hepatic fraction. The percentage of residual activity was calculated for four inhibitors at concentrations of 10⁻³, 10⁻⁴, and 10⁻⁵ M (pyrazole, 4-methylpyrazole, cimetidine, theophylline) and methylene blue at concentrations 10⁻⁴ and 10⁻⁵ M. Our results have shown that the best inhibitor, cimetidine, decreased oxidation of 0.1 M and 0.05 M methanol to 24 and 29% respectively at a drug concentration of 1 mM. Reaction with 0.1 M ethylene glycol as the ADH substrate was blocked by the same substances and 4-methylpyrazole was found to be a highly effective inhibitor.

Keywords: Key words Human alcohol dehydrogenase; Pyrazole; 4-Methylpyrazole; Cimetidine; Methylene blue

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Archives of Toxicology (v.72, #9)