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Archives of Toxicology (v.69, #1)

Molecular dosimetry of DNA adducts in rainbow trout (Oncorhynchus mykiss) exposed to benzo(a)pyrene by different routes by David Potter; Thomas M. Clarius; Alan S. Wright; William P. Watson (pp. 1-7).

Farm raised rainbow trout (Oncorhynchus mykiss) were exposed by various routes to benzo(a)pyrene (BP) as a representative carcinogenic polycyclic aromatic hydrocarbon (PAH). Following exposure of fish to the chemical by intraperitoneal (i.p.) injection, ³²P-postlabelling studies indicated that non-feral trout were relatively resistant to the formation of BP-DNA adducts in liver. No adducts were detected in fish exposed to single doses (20 mg/kg) of BP. Multiple exposures (e.g. 2×25 mg/kg) were necessary in order for adducts to be detected, indicating that induction of the metabolising enzymes required for the bioactivation of BP is necessary. These studies provided reference information on DNA adducts for comparison with data from subsequent experiments at environmentally realistic low level exposures. Two types of low level aquatic exposure were carried out. The first procedure exposed fish for 30 days to a nominally constant low level (1.2 and 0.4 μg/l) of a homogeneous dispersion of BP in water, to simulate low level aquatic environmental exposures. Following ³²P-postlabelling analysis of the liver DNA of exposed fish, BP-DNA adducts were not detected. In the second procedure, fish were exposed to a constant low level of BP (ca. 0.5 μg/l) for 15 days then to a pulse (60 μg/l) which was allowed to naturally decline (to ca. 2 μg/l) during a further 15 days. Following this exposure, significant levels of BP-DNA adducts were detected in livers of trout. The effect of dietary exposures was investigated by feeding trout a diet containing either 58 μg or 288 μg BP per day for 6 days, equivalent to total doses of 43 mg/kg and 216 mg/kg. In both cases BP-DNA adducts were detected in livers of exposed fish. The results provide useful information on the types of exposures to PAHs which may pose a genotoxic risk to fish in the environment.

Keywords: Benzo(a)pyrene; DNA adduct; Liver Rainbow trout (Oncorhynchus mykiss)

Formation and removal of DNA adducts in Fischer-344 rats exposed to 2,4-diaminotoluene by David K. La; John R. Froines (pp. 8-13).

³²P-Postlabeling was used to examine DNA adduct formation and removal in Fischer-344 rats exposed to the animal carcinogen 2,4-diaminotoluene (DAT). Adduct formation and persistence were compared between target (liver and mammary gland) and non-target organs (kidney and lung) to determine if possible differences could explain the observed organ specificity of DAT induced carcinogenesis. The effects of different exposure conditions on genesis. The effects of different exposure conditions on DNA adduct formation and removal were also examined by varying the concentration and frequency of compound administration. DAT produced three distinct DNA adducts. Among the organs examined, DNA binding was highest in the liver, with levels approximately 10 times greater than that of the mammary gland and up to 50 times greater than of the two nontarget sites. Despite the large differences in the initial extent of adduct formation, the persistence of adducts among sites was not significantly different. In the liver, there were dose-dependent differences in DNA adduct formation, but adduct removal following different dosages did not vary significantly. The effects of multiple administration on DNA adduct formation and removal were examined by treating rats with 5 mg/kg DAT daily for 10 consecutive days. Adduct yields from multiple treatment were greater than from a single 50 mg/kg exposure. The persistence of adducts following multiple treatment was also greater than after an equivalent single exposure. The results demonstrated organ-specific and dose-dependent differences in initial extent of DNA adduct formation, but no differences in adduct persistence. However, the results did suggest that adduct formation and persistence may change with repeated administration of DAT.

Keywords: DNA adducts; DNA adduct persistence 2,4-Diaminotoluene; ³²P-Postlabeling

Metabolism of a glucuronide conjugate of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in rats by Sunday Ene-ojo Atawodi; Kathrin Michelsen; Elmar Richter (pp. 14-17).

Besides 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), [4-(methylnitrosamino)-1-(3-pyridyl)but-1-yl]-β-O-d-glucosiduronic acid (NNAL-Glu) is another important metabolite of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) which has been detected in the urine of tobacco users and non-smokers heavily exposed to sidestream cigarette smoke. In order to evaluate the toxicological significance of NNAL-Glu formation and excretion, the metabolism of [5-³H]-NNAL-Glu was studied in rats. Five male F344 rats were administered 3.7 mg/kg [5-³H]-NNAL-Glu by i.v. injection and the metabolites in urine analysed by HPLC. More than 90% of the radioactivity was excreted in urine within the first 24 h. Unchanged NNAL-Glu accounted for 81.2±3.1% of the total radioactivity; the remaining part of the dose appears to be deconjugated resulting in the urinary excretion of NNAL (3.6±1.7%) and its α-hydroxylation (11.5±2.2%) and N-oxidation (3.6±1.6%) products. The presence of α-hydroxylation products of NNAL-Glu in urine suggests that this NNK metabolite may be activated in vivo to carcinogenic intermediates.

Keywords: Tobacco-specific nitrosamine 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone [4-(Methylnitrosamino)-1-(3-pyridyl)but-1-yl]-β-O-d-glucosiduronic acid; Rat; Urine; Metabolism

Dose and route dependency of metabolism and toxicity of chloroform in ethanol-treated rats by Pei -Yu Wang; Takashi Kaneko; Hiroshi Tsukada; Akio Sato (pp. 18-23).

The effects of a single dose of ethanol on the metabolism and toxicity of chloroform administered to rats per os (p.o.), intraperitoneally (i.p.), or by inhalation (inh) at different doses were investigated. Rats that had been given either ethanol (2 g/kg) or vehicle (water) alone at 4 p.m. on the previous day were challenged with chloroform at 10 a.m. p.o. (0, 0.1, 0.2, or 0.4 g/kg), i.p. (0, 0.1, 0.2, or 0.4 g/kg), or inh (for 6 h each at 0, 50, 100, or 500 ppm). The ethanol treatment, which had no influence on the intake of food and water, increased chloroform metabolism in vitro about 1.5-fold with no significant influence on liver glutathione content. The treatment had a dose-dependent effect o nthe metabolism and toxicity of chloroform, and the effect differed depending on the route of administration. Compared at the same dose level, the area under the curve (AUC) of blood chloroform concentration was invariably smaller following p.o. than i.p. administration. In accordance with this, chloroform administered p.o. caused more deleterious hepatic damage than the same amount of chloroform administered i.p. Although ethanol treatment had no significant influence on the AUC at any dose by any route of administration, the toxicity of p.o.-administered chloroform was significantly higher in ethanol-treated rats than in control rats at a dose as low as 0.1 g/kg, whereas no significant difference was observed in toxicity between both groups of rats at such a low dose administered i.p. When rats were exposed inh to air containing chloroform vapor, ethanol consumption had no effect on hepatotoxicity until the exposure concentration was raised to 500 ppm, a finding which suggests that a single dose of ethanol (2 g/kg) affects the toxicokinetics of inhaled chloroform in rats only at a concentration as high as 500 ppm.

Keywords: Chloroform; Toxicokinetics; Ethanol Enzyme induction; Dose; Route of administration

Allyl alcohol cytotoxicity in isolated rat hepatocytes: mechanism of cell death does not involve an early rise in cytosolic free calcium by Lora E. Rikans; Yong Cai; K. Roger Hornbrook (pp. 24-29).

We examined the effect of a toxic concentration of allyl alcohol (0.5 mM) on intracellular calcium concentrations in isolated rat hepatocytes. An increase in phosphorylase a activity was evident in the hepatocytes after 30 min of incubation with allyl alcohol, suggesting that the toxicant may produce an early rise in cytosolic free calcium. The increase in phosphorylase a activity was not reversed by the addition of dithiothreitol (DTT), a sulfhydryl compound that reverses the events that initiate cell killing by allyl alcohol. When intracellular calcium concentrations were measured directly, using fura-2 as the calcium indicator, there was no effect of allyl alcohol on cytosolic free calcium during the first 60 min of exposure, a critical period for development of irreversible damage. Incubation with allyl alcohol did not interfere with the measurement of intracellular calcium. The increases in cytosolic free calcium produced by phenylephrine or ATP were similar to those reported by others and not affected by the presence of allyl alcohol. The results from this study demonstrate that increased cytosolic free calcium is not essential for allyl alcohol-induced cytotoxicity to isolated rat hepatocytes.

Keywords: Allyl alcohol; Isolated hepatocytes; Calcium; Phosphorylase a

Comparison of hepatotoxicity caused by mono-, di- and tributyltin compounds in mice by Shunji Ueno; Nobuyuki Susa; Yoshinori Furukawa; Masayasu Sugiyama (pp. 30-34).

The in vivo induction of hepatotoxicity, as evaluated by the activity of ornithine carbamyl transferase in serum, was investigated in mice administered orally with the following three butyltin compounds: tributyltin chloride (TBTC), dibutyltin dichloride (DBTC) and monobutyltin trichloride (MBTC). The minimal concentrations of TBTC and DBTC that caused hepatotoxicity at 24 h after oral administration were 180 μmol and 60 μmol/kg, respectively, while MBTC did not induce liver injury even at 7000 μmol/kg. Additionally, when the administered doses were equivalent (180 μmol/kg), a time course (3–96 h) study revealed that the hepatotoxicity of TBTC and DBTC appeared at 24 and 12 h, respectively, but that MBTC showed no hepatotoxicity even at 96 h. The amounts of Sn excreted into urine for 4 days were 1.5 fold greater with TBTC than with DBTC treatment and were lowest in MBTC group. Similarly, the total liver Sn content was 2- to 5-fold greater in the TBTC group than in the DBTC group whereas the liver Sn content in the MBTC treatment showed the lowest value throughout the 3- to 96-h period. Thus, the non-hepatotoxicity of MBTC may be due either to low absorption through the digestive tract of mice or to the low levels of Sn in liver; however, the level of Sn in liver was not associated with the induction of hepatotoxicity by TBTC and DBTC. The analysis of metabolites of TBTC (180 μmol/kg) and DBTC (60 μmol/kg) at equivalent hepatotoxicity showed that the main tin compounds in the liver after the administration of TBTC were dibutyltin and monobutyltin as well as inorganic tin compounds, while most (>78%) of the total tin compounds in the liver of mice treated with DBTC was in the form of dibutyltin. In addition, the levels of monobutyltin and inorganic tin compounds in the livers of mice treated with TBTC were greater than those with DBTC, but the levels of dibutyltin did not differ significantly between TBTC and DBTC. The levels of lipid peroxidation (LPO) and hepatic glutathione (GSH) content in the liver showed a transitory increase after the administration of MBTC and TBTC, respectively. These results suggest that DBTC is more hepatotoxic than TBTC, and that dibutyltin inside the cells may be the main form of tin which is responsible for induction of hepatotoxicity following in vivo administration of TBTC and DBTC. The generation of free radical species, as evaluated by LPO and GSH levels, may not be associated with the hepatotoxicity caused by butyltin compounds.

Keywords: Butyltin compound; Hepatotoxicity; Lipid peroxidation; Glutathione

Effect of various antidotes on the biliary and intestinal excretion of arsenic in situ and into the feces in vivo in guinea-pigs after injection of As₂O₃ by Franz-Xaver Reichl; Helmut Kreppel; Ladislaus Szinicz; Harald Mückter; Burckhard Fichtl; Wolfgang Forth (pp. 35-38).

The effect of various antidotes on the excretion of arsenic into the feces in vivo and on the biliary and enteric excretion in situ was investigated on segments of jejunum and colon in anesthetized guinea-pigs using the pendular perfusion technique, according to Henning and Forth (1982). In the in situ experiments guinea-pigs received As₂O₃ (0.02 mmol As(III)/kg) and 30 min later, British-Anti-Lewisite (BAL), dimercaptopropanesulfonic acid (DMPS), dimercaptosuccinic acid (DMSA) or 2,3-bis-(acetylthio)propanesulfonamide (BAPSA) (0.1 or 0.7 mmol/kg each) into the jugular vein. In the in vivo experiments guinea-pigs received As₂O₃ s. c. (same dose as above) and 30 min later the same antidotes (0.1 mmol/kg i.p.). The feces were collected for 24 h and the arsenic content measured. During the 60-min perfusion period the amount of arsenic excreted into the jejunum or colon was only 3% or 0.4% of the dose administered, respectively. Of the arsenic dose, 8% was found in the bile. None of the antidotes had an effect on the arsenic excretion into the jejunum or colon. No change in biliary excretion was found in animals treated with BAL, 0.1 or 0.7 mmol/kg, respectively. DMSA, BAPSA or DMPS, 0.1 mmol/kg, increased the biliary excretion of arsenic to 14, 33, or 43% of the dose administered and after 0.7 mmol/kg to 29, 37, or 42%, respectively. Furthermore, a significant increase (P>0.05) was found for the bile/blood concentration ratio in the following order: control

Keywords: Arsenic excretion; Arsenic antidotes; Bile; Jejunum; Colon; Feces; Guinea-pigs

Comparative toxicity of methyl isocyanate and its hydrolytic derivatives in rats by K. Jeevaratnam; S. Sriramachari (pp. 39-44).

The present study describes the acute histopathological changes induced by methyl isocyanate (MIC) in the lungs of rats at 24 h after a single exposure to varied concentrations/doses of MIC by inhalation and subcutaneous (s.c.) routes and also delineates the effects due to the hydrolytic derivatives of MIC, viz., methylamine (MA) and N,N′-dimethyl urea (DMU). MIC, either inhaled or administered s.c., resulted in a wide range and extent of histopathological changes in the lungs, proportional to the exposure concentration/dose. The salient, effects of inhaled MIC are acute necrotizing bronchitis of the entire respiratory tract accompanied by varying degrees of confluent congestion, hyperemia and interstitial and intra-alveolar edema, while MIC administered s.c. led to prominent vascular endothelial damage, congestion and severe interstitial pneumonitis with apparently normal bronchial epithelium; and intra-alveolar edema only with the high dose. The only noteworthy lesion produced by MA and DMU (to some extent) was interstitial pneumonitis, suggesting their possible involvement in the subsequent inflammatory response of MIC. Except, for the endothelial changes, the overall spectrum of the histopathological lesions is quite comparable to those observed in the lungs of Bhopal victims during the acute phase.

Keywords: Methyl isocyanate toxicity; Histopathology; Lungs; Methylamine toxicity N,N′-Dimethyl urea toxicity; Rats

Comparative toxicity of methyl isocyanate and its hydrolytic derivatives in rats by S. Sriramachari; K. Jeevaratnam (pp. 45-51).

This paper describes the long-term (subacute and chronic) histopathological effects in the lungs of rats subjected to a single exposure to methyl isocyanate (MIC) by both the inhalation and subcutaneous (s.c.) routes as well as the role of methylamine (MA) and N,N′-dimethylurea (DMU), the hydrolytic derivatives of MIC in eliciting the observed changes. At the subacute phase, the intraalveolar and interstitial edema were prominent only in the inhalation group as against the more pronounced inflammatory response in the s.c. route. With the progress of time the evolution of lesions appeared to be similar, culminating in the development of significant interstitial pneumonitis and fibrosis. MA, one of the hydrolytic derivatives of MIC, also caused interstitial pneumonitis progressing to fibrosis, albeit to a lesser extent than MIC, indicating its contribution to the long-term pulmonary damage. The diffuse interstitial pulmonary fibrosis observed at 10 weeks after a single exposure to MIC by either route is of greater significance in the context of the occurrence of pulmonary fibrosis in the late autopsies of Bhopal gas victims and also clinical sequelae in some of the survivors.

Keywords: Methyl isocyanate toxicity; Methylamine toxicity; Pulmonary pathology; Long-term effects; Fibrosis; Rats

Assessment of the labelling index of cohorts of the pancreatic islet cell population in phenobarbitone-treated male rats using a double immunohistochemical technique for 5-bromo-2′-deoxyuridine and pancreatic hormones by Huw B. Jones; Stephen J. Harbottle; Alison L. Bowdler (pp. 52-58).

A previous study demonstrated that administration of phenobarbitone to male AP Wistar rats for up to 7 days caused alterations in labelling indices (LIs) in several different tissues (including a reduction of the endocrine pancreas population LI) as determined by immunohistochemical visualisation of 5-bromo-2′-deoxyuridine (BrdU) incorporation into S-phase nuclei. The primary objective of this study was to determine whether treatment with phenobarbitone influenced the replicative states of specific cohorts of the islet (of Langerhans) cell population or generated a uniform depression of LI. Quantitation of the LIs of individual islet cell cohorts was achieved by utilisation of a dual immunohistochemical staining method for BrdU and islet hormones (insulin, glucagon and somatostain) using a sequential peroxidase anti-peroxidase (PAP)/alkaline phosphatase anti-alkaline phosphatase (APAAP) method employing diaminobenzidine and New Fuchsin chromogens, respectively. We observed reductions, increases and no change in LIs of insulin-, glucagon- and somatostatin-positive cells, respectively. We conclude that the decreased LI of the insulin-positive cohort was not countered entirely by the LI increase in the glucagon-positive cohort due to the larger size of the former. Furthermore, the effects of phenobarbitone treatment are not manifested generally in the islet cell population but in the insulin- and glucagon-positive cohorts only. The causation of these effects is unknown but is likely to be due to enhanced carbohydrate and hormone metabolism. We believe that the visualisation and quantitation of replicating cells in specific hormone-positive cohorts of the islet cell population provide opportunities for understanding the influence of xenobiotics and disease processes on pancreatic function.

Keywords: Cell proliferation; Pancreas; 5-Bromo-2′-deoxyuridine (BrdU); Immunohistochemistry; Hormones; Insulin; Glucagon; Somatostatin; Phenobarbitone

Time course of effects of tetraethoxysilane (TEOS) on the kidney and blood silicon concentration in mice by Hiroshi Nakashima (pp. 59-64).

To clarify the time course of toxicological effects of tetraethoxysilane [Si(OC₂H₅)₄, TEOS] on the kidney and the relationship between blood silicon levels (Si−B) and the effects, 250 mg/kg or 500 mg/kg TEOS was intraperitoneally administered to ten 5-week-old male ICR mice (SPF grade) in each group, and morphological and functional changes of the kidney were assessed at 12 h, 24 h, 3 days and 2 weeks after administration of TEOS. Injury to tubular epithelial cells was observed in mice killed 12 and 24 h after administration, and its severity increased with increasing dosage. The mean values of blood urea nitrogen exhibited dose-related increase in mice sacrificed 24 h after the administration. The concentrations of Si−B increased in order of the administered doses of TEOS, and then decreased steadily. The results of Si−B were consistent with the concept that renal toxicity of TEOS is mediated by siliceous compounds. The kidney was recovering from injury 3 days after administration, and had developed tubulointerstitial nephritis, which could be regarded as repaired lesion of acute injury, by 2 weeks after administration.

Keywords: Tetraethoxysilane; Semiconductor; Time course study; Renal toxicity; Blood silicon concentration

Embryotoxic effects of L-691,121, a class III antiarrhythmic agent, in rats by Yoshiki Ban; Reiko Konishi; Ken-ichi Kawana; Toshio Nakatsuka; Takaaki Fujii; J. M. Manson (pp. 65-71).

L-691, 121 is a class III antiarrhythmic agent which blocks potassium currents, leading to prolongation of cardiac potential and prevention of cardiac arrhythmia. In a developmental toxicity study in rats, there was a dose-dependent decrease in embryonic/fetal survival, and death of the entire litter was seen at an oral dose of 0.8 mg/kg per day. The critical period for embryolethality was determined as gestational days (GD) 10–13. In a study where females received 1 mg/kg on a critical day (GD 10 or 12) and were killed at 24-h intervals, a high embryonic mortality was seen at 72 h (GD 10 treatment) or 48 h (GD 12 treatment) after dosing. The surviving embryos had morphological abnormalities such as enlarged cardiac tube and pericardium, generalized edema, and hematoma. In order to investigate a possible mechanism for the embryolethality, GD 11 embryos were dissected from females at 4h after dosing of 1 mg/kg and incubated for 5 h in vitro. The embryonic heart rates were decreased for the first 2 h after incubation but tended to recover to control levels thereafter. When GD 11 embryos were incubated for 4 h with the drug, there were decreases in the heart rates during the entire observation period. In a washout study where the embryos were transferred to drug-free medium after 1-h exposure, decreased heart rates recovered to control levels. In GD 11 embryos cultured for 24 h with the drug, there were gross abnormalities that consisted of altered yolk sac and embryonic circulation, and enlargement of cardiac tube and pericardium similar to those seen in the preceding in vivo study. These results suggest that decreased preceding in vivo study. These results suggest that decreased heart rates, reduced yolk sac circulation and the associated morphological abnormalities induced by L-691,121 are related to the embryolethality in rats.

Keywords: Antiarrhythmia; Embryotoxicity; L-691,121 Rats

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Archives of Toxicology (v.69, #1)