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Archives of Toxicology (v.72, #11)

Nickel(II) inhibits the repair of O6-methylguanine in mammalian cells by Frank Iwitzki; Regina Schlepegrell; Uta Eichhorn; Bernd Kaina; Detmar Beyersmann; Andrea Hartwig (pp. 681-689).

Nickel compounds are widespread carcinogens, and although only weakly mutagenic, interfere with nucleotide excision repair and with the repair of oxidative DNA base modifications. In the present study we investigated the effect of nickel(II) on the induction and repair of O⁶-methylguanine and N7-methylguanine after treatment with N-methyl-N-nitrosourea (MNU). We applied Chinese hamster ovary cells stably transfected with human O⁶-methylguanine-DNA methyltransferase (MGMT) cDNA (CHO-AT), and compared the results with the MGMT-deficient parental cell line. As determined by high-performance liquid chromatography/electrochemical detection (HPLC/ECD), there was a slight but mostly not significant reduction in the formation of both types of DNA lesions by MNU in the presence of nickel(II). Although nickel(II) did not markedly affect the repair of N7-methylguanine, it decreased the repair of O⁶-methylguanine in a dose-dependent manner, starting at concentrations as low as 50 μM. While the MGMT protein level was not altered in the presence of nickel(II), the MGMT activity was diminished as demonstrated in cell extracts form nickel-treated cells. This repair inhibition was accompanied by an increase in MNU-induced cytotoxicity in nickel-treated CHO-AT cells but not in MGMT-deficient control cells. There is strong evidence that O⁶-methylguanine is involved in tumour formation after exposure to alkylating agents. Thus, the finding that nickel(II) inhibits the repair of this lesion could be of major importance for risk assessment in case of combined exposures at work places and in the general environment.

Keywords: Key words Nickel; O6-methylguanine; MGMT; repair; Mammalian

Evidence that the reactions of cadmium in the presence of metallothionein can produce hydroxyl radicals by Paul O'Brien; Henryk J. Salacinski (pp. 690-700).

A variety of Cu reconstituted metallothioneins (MTs) containing different amounts of copper ions together with Cd₇-MT free of copper were prepared and used in spin trapping experiments designed to show cadmium is not a Fenton active metal. A significant increase of the DMPO/·OH adduct was observed, with increased concentrations of the copper containing MTs, H₂O₂ enhanced DMPO/·OH adduct formation, catalase and the Cu (I) specific chelating agent bathocuproine, reduced DMPO/·OH adduct formation. These results suggest that Cu (I) and H₂O₂ both have important roles in the production of active species in these systems and cause DMPO/·OH formation. However, Cd₇-MT showed no ability to cause generation of DMPO adducts with H₂O₂ seeming to indicate cadmium is not a Fenton metal. To test this hypothesis further trapping studies were run with added sulphite and lipid peroxide using both commercial MT and Cd₇-MT since cadmium causes peroxidation in vivo. Commercial MT generates radicals with added sulphite and peroxide, Cd₇-MT does not, demonstrating that cadmium is not a Fenton metal. These results help to explain the oxidative damage to DNA observed in the presence of MT and cadmium in vitro.

Keywords: Key words Cadmium; Metallothionein; Hydroxyl radical; Lipid peroxidation; Non Fenton

Three cases of methylmercury intoxication which eluded correct diagnosis by Lászió Magos (pp. 701-705).

Three casual workers engaged in the production of mercuric acetate were admitted to hospital within 22 calendar days of each other, respectively 30, 48 and 5 days after their last working day. The workers served the same reactor in which elemental mercury was oxidized by peroxide and mercuric acetate was formed by the reaction of mercuric oxide with acetic acid. The diagnosis of mercury vapour intoxication of the first two patients was made 21 and 16 days after their admission when the third patient was admitted and hospitals were informed about their exposure. This diagnosis was made without considering: (a) that the observed signs were characteristic of methylmercury intoxication and are rarely present in mercury vapour intoxication; (b) the degree of deterioration after removal from exposure implicated methylmercury; (c) that blood mercury concentrations extrapolated to the last day at work were in the range, which had been associated with severe intoxication in the Iraq methylmercury epidemic; (d) at the time of the first blood mercury estimations the blood urinary mercury concentration ratios were 11.2, 5.4 and 2.4 while this ratio is below 0.5 in mercury vapour intoxication or in workers exposed to mercury vapour.

Keywords: Key words Methylmercury; Elemental mercury; Mercury in blood; Mercury in urine; Occupational exposure

Arsenobetaine is not a major metabolite of arsine gas in the rat by Jean-Pierre Buchet; Pietro Apostoli; Dominique Lison (pp. 706-710).

Many organisms can easily dispose of toxic inorganic arsenic species through gradual methylation of the element and further urinary excretion. In order to clarify the urinary excretion of arsenobetaine observed in a human case of intoxication by arsine, the capacity of highly methylated arsenical synthesis has been investigated in rats acutely exposed during 1 h to increasing concentrations of the same gas [4 to 80 mg AsH₃/m³]. Urinary metabolites of arsenic were determined with good agreement in two (Belgian and Italian) laboratories using two different analytical procedures. The sum of inorganic, mono- and dimethylated metabolites of arsenic in urine was shown to be related to the intensity of exposure to arsine. A biphasic relationship was observed: 1 h exposure to >60 mg AsH₃/m³ led to metabolite excretion which is roughly 10 times higher than for exposure levels below that limit, suggesting the saturation of a binding site reserve and the availability for metabolism of a greater proportion of the As absorbed above this threshold. Arsenobetaine production, if any, could only be detected when its presence in food was excluded; in addition, amounts appeared negligible and could be disregarded as a common arsenic metabolite in rats.

Keywords: Keywords Arsine gas; Metabolism; Arsenobetaine; Rat

Biological effects of the dihydroorotate dehydrogenase inhibitor polyporic acid, a toxic constituent of the mushroom Hapalopilus rutilans , in rats and humans by Jeanette Kraft; Siefgfried Bauer; Gerburg Keilhoff; Jürgen Miersch; Detlef Wend; Dagmar Riemann; Rolf Hirschelmann; Hans-Jürgen Holzhausen; Jürgen Langner (pp. 711-721).

Inhibitors of dihydroorotate dehydrogenase (DHO-DH), such as brequinar or leflunomide, have been intensively tested for their antitumour and immunomodulating effects. Polyporic acid (PA) from the mushroom Hapalopilus rutilans (H. r.) also is a DHO-DH inhibitor (50% inhibitory concn., IC₅₀, 10⁻⁴–10⁻³ M). As three people had been poisoned following ingestion of H. r. we wanted to investigate the effects of PA in rats and in cell cultures. Rats given PA via probang (100–800 mg/kg) within 24 h developed strongly reduced locomotor activity, depressed visual placing response and impaired wire manoeuvre. Laboratory investigation of blood revealed hepatorenal failure, metabolic acidosis as well as hypokalaemia and hypocalcaemia. All symptoms closely paralleled the effects seen in the poisoned people. Proliferation of cultured cells (including rat brain neurons and glia, fibroblasts, tumour cells) was depressed at 10⁻⁴–10⁻³ M PA. We conclude that the intoxication of people poisoned with H. r. is due to the high content of the DHO-DH inhibitor PA.

Keywords: Key words Polyporic acid; Dihydro orotate dehydrogenase; Hapalopilus rutilans; Brequinar; Leflunomide

Effect of lindane and heptachlor on δ-aminolaevulinate synthase and its regulation by María Cristina Taira; Leonor C. San Martín de Viale (pp. 722-730).

The effect of lindane and heptachlor on haem metabolism was studied with the aim to elucidate the mechanism of their porphyrinogenic action. The effects of these compounds on δ-aminolaevulinate synthase (ALA-S) and ferrochelatase were evaluated and the mechanism of increase of ALA-S activity was especially studied. The results indicated the following: (1) Lindane and heptachlor produced increases in ALA-S activity; this effect was dependent on the drug dose, the time of treatment, and the development of the animal, the maximum response being obtained prior to hatching. Lindane was observed to have a greater effect on ALA-S than heptachlor. In fact, when effects of lindane and heptachlor were compared we observed that lindane produced: (a) greater increases in ALA-S activity (six fold vs four fold), both with respect to dimethyl sulphoxide (DMSO) controls (3.8 ± 0.3 nmol ALA/g liver per h); (b) earlier ALA-S response (1.5 h vs 4 h); (c) responses at lower doses (0.3 mg/egg vs 1 mg/egg). (2) The increase in ALA-S activity produced by lindane or heptachlor is an induction and not an activation process since it depends on protein synthesis and the drugs per se have no effect. Thus, our results obtained from studies in ovo with actinomycin D and cycloheximide suggest that lindane is acting at the translational level while heptachlor interferes at the level of transcription. (3) The study of ALA-S subcellular distribution indicated no accumulation in the cytosol of DMSO controls and in the lindane or heptachlor treated embryos, neither of the chlorinated pesticides alter the normal subcellular distribution of this regulatory enzyme in the liver (4) Exogenous haem was able to prevent or decrease the induction of ALA-S elicited by both pesticides, thus showing that lindane or heptachlor-induced ALA-S respond to haem regulation. (5) Lindane had no effect on ferrochelatase activity at the doses and times assayed, but heptachlor decreased this enzyme activity. The porphyrinogenic mechanism of lindane and heptachlor is discussed on the basis of the present results.

Keywords: Key words Porphyria; Heptachlor; Lindane; δ-aminolaevulinate synthase (ALA-S); Ferrochelatase; Haem; Enzyme induction; Protein inhibitors; ALA-S subcellular distribution

Theophylline-induced mesenteric periarteritis in F344/N rats by Abraham Nyska; Ronald A. Herbert; Po C. Chan; Joseph K. Haseman; James R. Hailey (pp. 731-737).

The toxicity and carcinogenic potential of theophylline (an alkaloid bronchodilator drug) was investigated in male and female F344/N rats in 16-day, 14-week, and 2-year gavage and feeding studies. In 16-day studies, rats were fed diets containing 0, 500, 1000, 2000, 4000, and 8000 ppm of theophylline or given 0, 12.5 (twice daily), 25 (once daily), 50 (once daily), 50 (twice daily), 100 (once daily), 200 (once daily), 200 (twice daily), and 400 (once daily) mg theophylline/kg body weight in corn oil by gavage. In 14-week studies, rats were fed diets containing 0, 1000, 2000, and 4000 ppm theophylline or given 0, 37.5, 75, and 150 mg/kg body weight theophylline in corn oil by gavage. In 2-year gavage studies, rats were given 0, 7.5, 25, and 75 mg/kg body weight in corn oil. In 16-day gavage studies, treatment-related periarteritis occurred in arteries of the pancreas and adjacent to the mesenteric lymph nodes of early death male and female rats given 400 mg/kg once daily. In the 14-week studies, treatment-related periarteritis occurred at similar sites and in male rats exposed to 75 and 150 mg/kg, and in all exposed female rats (gavage studies), in females exposed to 1000 ppm, and in both sexes exposed to 2000 and 4000 ppm (feeding studies). In the 2-year study, chronic periarteritis was significantly increased only in the males receiving 75 mg/kg of theophylline. The adventitia, media and intima of medium- and large-sized mesenteric arteries were involved. Similar to other vasodilator chemicals, the pathogenesis of theophylline-induced vascular lesions may be a consequence of hemodynamic changes induced in the vascular wall.

Keywords: Key words Theophylline; Alkaloid; Pharmaceutical; Bronchodilator; Arteritis

Chinese herbs nephropathy-associated slimming regimen induces tumours in the forestomach but no interstitial nephropathy in rats by Jean-Pierre Cosyns; Rose-Marie Goebbels; Vinciane Liberton; Heinz H. Schmeiser; Christian A. Bieler; Alfred M. Bernard (pp. 738-743).

Chinese herbs nephropathy (CHN), a rapidly progressive interstitial fibrosis of the kidney, has been described in approximately 100 young Belgian women who had followed a slimming regimen containing some Chinese herbs. In 4 patients multifocal transitional cell carcinomas (TCC) were observed. Aristolochic acid (AA), suspected as the causal factor of CHN, is a well known carcinogen but its ability to induce fibrosis has never been demonstrated. The objective of this study was to evaluate the latter using doses of AA, durations of intoxication and delays of sacrifice known to yield tumours in rats. We also tested the hypothesis that a possible fibrogenic role of AA was enhanced by the other components of the slimming regimen. Male and female rats were treated orally with 10 mg isolated AA/kg per day for 5 days/week, or with approximately 0.15 mg AA/kg per day 5 days/week contained in the herbal powder together with the other components prescribed in the slimming pills for 3 months. The animals were killed respectively 3 and 11 months later. At sacrifice, animals in both groups had developed the expected tumours but not fibrosis of the renal interstitium. Whether the fibrotic response observed in man is due to species and/or strain related differences in the response to AA or to other factors, remains to be determined. Interestingly, despite the addition of fenfluramine and diethylpropion, two drugs incriminated in the development of valvular heart disease, no cardiac abnormalities were observed.

Keywords: Key words Aristolochic acid; Chinese herbs; Carcinogenicity; Toxicology

Mechanistic study on liver tumor promoting effects of piperonyl butoxide in rats by Hideaki Okamiya; Kunitoshi Mitsumori; Hiroshi Onodera; Seiichi Ito; Takayoshi Imazawa; Kazuo Yasuhara; Michihito Takahashi (pp. 744-750).

Piperonyl butoxide, alpha-[2-(2-butoxyethoxy)ethoxy]-4,5-methylenedioxy-2-propyltoluene, is a widely used pesticide-synergist. Recently, results were reported indicating that piperonyl butoxide is a hepatocarcinogen in rat. Since the underlying mechanism was not elucidated, we examined the effects on rat liver cells in detail. For this purpose male F344 rats were administered piperonyl butoxide mixed in the diet at concentrations of 0 (negative control), 0.05, 0.2 or 2% for 2 days, 1, 2, and 4 weeks. As a positive control, phenobarbital was administered to rats for up to 4 weeks as a 0.1% solution in the drinking water. Increased liver weight, centrilobular hepatocellular hypertrophy due to increased smooth endoplasmic reticulum, decreased numbers and areas of connexin 32-positive spots per hepatocyte, and increased cell proliferation were observed in rats treated with 0.2 and 2% piperonyl butoxide. Similar results were obtained for 0.1% phenobarbital treated rats. Hepatocellular necrosis suggestive of hepatotoxicity was also observed in the 2% piperonyl butoxide group. These results indicate that the promoting mechanism of piperonyl butoxide in hepatocarcinogenesis is similar to that of phenobarbital, involving an ability to induce CYP isoenzymes and inhibit gap junctional intercellular communication. In addition, increased cell proliferation following hepatocellular necrosis may also play a role at high doses.

Keywords: Key words Piperonyl butoxide; Phenobarbital; Hepatocarcinogenesis promoting mechanism; Gap junctional intercellular communication; Cell proliferating activity

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Archives of Toxicology (v.72, #11)