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Archives of Toxicology (v.69, #10)

The international validation study of the acute toxic class method (oral) by E. Schlede; U. Mischke; W. Diener; D. Kayser (pp. 659-670).

An alternative to the oral LD₅₀ test, the acute toxic class (ATC) method (oral), was validated with 20 substances in an international collaborative study with nine laboratories in five countries. The ATC method is a stepwise procedure with the use of three animals per step. It has been designed with three fixed doses (25, 200 and 2000 mg/kg). In general, this testing is sufficient for allocation to the toxicity classes of the majority of the international classification systems currently in use. The selection of testing at additional fixed doses (5, 50 and 500 mg/kg) may be considered if further refinement is necessary or for specific allocation to those international classification systems with a cut-off value of 5 mg/kg. On average, two to four steps are necessary to complete a test. With the ATC method substances can be ranked in a similar or even better manner than with an LD₅₀ test but it uses up to 90% fewer animals, the average being 70% fewer. This also results in substantially fewer moribund/dead animals. The ATC method is based on biometric evaluations that, together with the experimental results, demonstrate that this method is a sensitive and reliable alternative to the LD₅₀ test.

Keywords: Acute toxic class method (oral); Alternative; LD₅₀ test; Animal welfare; International classification systems

An approach for evaluating the respiratory irritation of mixtures: application to metalworking fluids by M. M. Schaper; K. A. Detwiler-Okabayashi (pp. 671-676).

Recently, the sensory and pulmonary irritating properties of ten metalworking fluids (MWF) were assessed using a mouse bioassay. Relative potency of the MWFs was estimated, but it was not possible to identify the component(s) responsible for the the respiratory irritation induced by each MWF. One of the ten fluids, MWF “E”, produced sensory and pulmonary irritation in mice, and it was of moderate potency in comparison to the other nine MWFs. MWF “E” had three major components: tall oil fatty acids (TOFA), sodium sulfonate (SA), and paraffinic oil (PO). In the present study, the sensory and pulmonary irritating properties of these individual components of MWF “E” were evaluated. Mixtures of the three components were also prepared and similarly evaluated. This analysis revealed that the sensory irritation from MWF “E” was largely due to TOFA, whereas SA produced the pulmonary irritation observed with MWF “E”. Both TOFA and SA were more potent irritants than was MWF “E”, and the potency of TOFA and/or SA was diminished through combination with PO. There was no evidence of synergism of the components when combined to form MWF “E”. This approach for identifying the biologically “active” component(s) in a mixture should be useful for other MWFs. Furthermore, the approach should be easily adapted for other applications involving concerns with mixtures.

Keywords: Metalworking fluids; Pulmonary irritation; Mice; Mixtures

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on tryptophan and glucose homeostasis in the most TCDD-susceptible and the most TCDD-resistant species, guinea pigs and hamsters by Mikko Unkila; Marjatta Ruotsalainen; Raimo Pohjanvirta; Matti Viluksela; Ewen MacDonald; Jouni T. Tuomisto; Karl Rozman; Jouko Tuomisto (pp. 677-683).

We have previously reported that in rats 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) lethality is associated (although not necessarily causally) with changes in brain serotonin (5-HT) metabolism. In the present study, we have examined whether this holds for other species by comparing the effect of TCDD in the most TCDD-susceptible and the most TCDD-resistant species, guinea pigs and hamsters, respectively. Body weight gain of guinea pigs exposed to TCDD (0.3–2.7 μg/kg) diminished dose dependently, while the effect was marginal in hamsters (900–4600 μg/kg). Brain 5-hydroxyindoleacetic acid (the main metabolite of brain 5-HT), brain tryptophan (the precursor amino acid of 5-HT), and plasma free and total tryptophan were not affected at any dose in guinea pigs. In contrast, 4 days after exposure, the levels of plasma free and total tryptophan were consistently increased in hamsters. These, as well as brain tryptophan, were still elevated 10 days after exposure. TCDD did not affect plasma glucose level in either species. Liver glycogen was decreased in a dose-dependent manner in TCDD-treated guinea pigs as well as in their pair-fed controls on day 10. There was no change in liver glycogen in hamsters. The activity of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase was only depressed in hamsters by all doses of TCDD. We conclude that changes in tryptophan metabolism or in carbohydrate homeostasis cannot explain the wide interspecies differences in susceptibility to the acute lethality of TCDD, although they may correlate with some aspects of its toxicity in certain species.

Keywords: 2,3,7,8-Tetrachlorodibenzo-p-dioxin; Species differences; Acute toxicity; Serotonin; Tryptophan; Gluconeogenesis

Aspirin-like drugs can protect human T lymphocytes against benzoquinone cytotoxicity: by Eliezer Flescher; Carroll A. Snyder (pp. 684-689).

Benzene toxicity towards lymphocytes is thought to be mediated by metabolities of benzene including benzoquinone (BQ). NAD(P)H:quinone reductase (QR) is known to protect against BQ toxicity. The expression of the QR gene is regulated by the transcription factor AP-1. We had previously found that aspirin-like drugs (ALD) induce AP-1 in human T lymphocytes. It was therefore hypothesized that ALD would protect lymphocytes against BQ toxicity by inducing QR. Molt-4 cells (M4), a human T lymphocyte cell line, were incubated with different concentrations of two ALD, flurbiprofen and sodium diclofenac, and then exposed to BQ. Toxicity was measured by viability (trypan blue exclusion). Both drugs protected the cells against BQ cytotoxicity in a dose-dependent manner, e.g., sodium diclofenac at 15 μM reduced the fraction of BQ-treated dead cells by 70%. ALDs induced QR activity in the M4 cells in the same range of concentrations that protected the cells against BQ toxicity. The protective effect of ALD was significantly reduced by dicoumarol, a QR-specific inhibitor. Since human T cells and T cell lines do not metabolize arachidonic acid, our data suggest that ALD can protect human T lymphocytes against a metabolite of benzene by induction of QR activity.

Keywords: Non-steroidal anti-inflammatory drugs; Benzene; AP-1; Benzoquinone; NAD(P)H:quinone reductase

Comparative effects of two dithiocarbamates disulfiram and thiram, on adrenal catecholamine content and on plasma dopamine-β-hydroxylase activity by Stefano Caroldi; Paola De Paris (pp. 690-693).

Both disulfiram (tetraethylthiuram disulfide), an alcohol aversive drug, and thiram (tetramethylthiuram disulfide), a widely used pesticide, significantly increased the dopamine pool in the adrenal glands of dosed rats. The dopamine increase was detectable within 4 h of oral dosing with 100 mg/kg of either dithiocarbamate and peaked 24 h later at 10 times control values. In control rats the dopamine turnover was 0.51 h⁻¹ as calculated by the assumed first order decline of dopamine after a single injection of α-methyl-p-tyrosine (α-MT, 400 mg/kg i.p.) resulting in a dopamine-β-hydroxylase (DBH) activity of 0.73 nmol/h per pair of adrenals. In the adrenals of rats pretreated with thiram and then injected with α-MT, the adrenal dopamine content did not significantly decline, indicating that thiram reduced the conversion of dopamine to noradrenaline, eventually leading to the observed dopamine increase. Plasma DBH activity was significantly reduced 4 h and 24 h after dosing with thiram, but was unchanged after treatment with disulfiram. The determination of plasma DBH activity could be a marker to monitor the effect of thiram on catecholamine metabolism in occupationally exposed workers but not that of disulfiram in abstinent alcoholics.

Keywords: Thiram; Disulfiram; Dopamine-β-hydroxylase; Adrenal medulla

Effect of methyl isocyanate on rabbit cardiac Na⁺, K⁺-ATPase by K. Jeevaratnam (pp. 694-697).

The present study describes the effect of methyl isocyanate (MIC) on rabbit cardiac microsomal Na⁺, K⁺-ATPase. Addition of MIC in vitro resulted in dose-dependent inhibition of Na⁺, K⁺-ATPase, Mg²⁺-ATPase and K⁺-activated p-nitrophenyl phosphatase (K⁺-PNPPase). Activation of Na⁺, K⁺-ATPase by ATP in the presence of MIC showed a decrease in V_max with no change in K_m. Similarly, activation of K⁺ PNPPase by PNPP in the presence of MIC showed a decrease in V_max with no change in K_m. The circular dichroism spectral studies revealed that MIC interaction with Na⁺, K⁺-ATPase led to a conformation of the protein wherein the substrates Na⁺ and K⁺ were no longer able to bind at the Na⁺- and K⁺-activation sites. The data suggest that the inhibition of Na⁺, K⁺-ATPase was non-competitive and occurred by interference with the dephosphorylation of the enzyme-phosphoryl complex.

Keywords: Methyl isocyanate toxicity; Na⁺, K⁺-ATPase inhibition; Circular dichroism of Na⁺, K⁺-ATPase; Kinetics; Rabbit myocardium

Characterization of the arteritis induced by infusion of rats with UK-61,260, an inodilator, for 24 h by Gilles Hanton; Jean -Loic Le Net; Bernard Ruty; Bernard Leblanc (pp. 698-704).

Administration of fenoldopam mesylate (FM), a dopaminergic agonist, or of cyclic cAMP phosphodiesterase inhibitors (PDE III), for example theophylline and caffeine, induces arteritis in the rat. In this study we characterized the arteritis induced by UK-61,260, an investigational inotropic agent with vasodilatory properties which displays an inhibitory action on cyclic AMP phosphodiesterase, in comparison with lesions induced by FM. The compounds were administered to Sprague-Dawley rats by intravenous infusion over 24 h (FM and UK-61,260), orally or subcutaneously (UK-61,260); the rats were killed and necropsied for pathological examination at various times between 0 h and 28 days post-infusion. Infusion of UK-61,260 at doses of 100, 300 or 400 mg/kg produced arteritis mainly in the mesenteric arteries and occasionally in the renal, pancreatic, gastric and coronary arteries. There were no arterial lesions after infusion of 30 mg/kg, or after administration of 30, 100 or 200 mg/kg per day subcutaneously for 7 days, or after acute administration of 100, 300, 400 or 600 mg/kg orally. Infusion of rats with 72 or 144 mg/kg FM produced arteritis over a wider range of tissues than did UK-61,260. However, the arterial lesions produced by infusion of either drug have the same initial aspect and a similar evolution with time. Immediately after the end of the infusion, minimal necrosis and haemorrhage occurred in the media only, without involvement of the endothelium or the perivascular space. This indicates that the media of the artery is the primary site of injury. The lesions seen 1 and 3 days post-infusion were characterized by severe medial necrosis and haemorrhage with perivas cular acute inflammation and appeared macroscopically as haemorrhagic spots on the vessels. On days 7, 14 and 28 post-infusion, no medial necrosis or haemorrhage were present, while perivascular chronic inflammation and moderate smooth muscle hyperplasia were seen. It appeared therefore, that the lesions underwent repair in 28 days, but footprints of the damage were still present 28 days post-infusion. The similarity between arteritis induced in rats by fenoldopam or by UK-61,260, at doses inducing PDE III inhibition, is consistent with the view that they have a similar pathogenesis. In our view it is probable that these pharmacologically and chemically distinct drugs trigger an increase in intracellular levels of cAMP which in turn triggers vascular damage. The arterial changes observed in the current study after acute administration may explain the increased incidence of polyarteritis nodosa occurring in long term toxicity studies with FM or PDE III inhibitors.

Keywords: Phosphodiesterase inhibitor; Dopaminergic agonist; Arteritis; Medial necrosis; Vascular haemorrhage; Fenoldopam; UK-61,260

Triphenylphosphite neuropathy in hens by F. Fioroni; A. Moretto; M. Lotti (pp. 705-711).

Single doses of triphenyl phosphite (TPP), a triester of trivalent phosphorus, cause ataxia and paralysis in hens. Characteristics of neurotoxicity were described as somewhat different from organophosphate induced delayed polyneuropathy (OPIDP), which is caused by triesters of pentavalent phosphorus. The onset of TPP neuropathy was reported to occur earlier than that of OPIDP (5–10 versus 7–14 days after dosing, respectively), and chromatolysis, neuronal necrosis and lesions in certain areas of the brain were found in TPP neuropathy only. Pretreatment with phenylmethanesulfonyl fluoride (PMSF) protects from OPIDP, but it either partially protected from effects of low doses or exacerbated those of higher doses of TPP. In order to account for these differences with OPIDP, it was suggested that TPP neuropathy results from the combination of two independent mechanisms of toxicity: typical OPIDP due to inhibition of neuropathy target esterase (NTE) plus a second neurotoxicity related with other target(s). We explored TPP neuropathy in the hen with attention to the phenomena of promotion and protection which are both caused by PMSF when given in combination with typical neuropathic OPs. When PMSF is given before neuropathic OPs it protects from OPIDP; when given afterwards it exaggerates OPIDP. The former effect is due to interactions with NTE, the latter to interactions with an unknown site. The time course of NTE reappearance after TPP (60 or 90 mg/kg i.v.) inhibition showed a longer half-life when compared to that after PMSF (30 mg/kg s.c.) (10–15 versus 4–6 days, respectively). The clinical signs of TPP neuropathy (60 or 90 mg/kg i.v.) were similar to those observed in OPIDP, appeared 7–12 days after treatment, correlated with more than 70% NTE inhibition/aging and were preceded by a reduction of retrograde axonal transport in sciatic nerve of hens. TPP (60 mg/kg i.v.) neuropathy was promoted by PMSF (120 mg/kg s.c.) given up to 12 days afterwards and was partially protected by PMSF (10–120 mg/kg s.c.) when given 24 h before TPP (60 or 90 mg/kg i.v.). The previously reported early onset of TPP neuropathy might be related to the higher dose used in those experiments and to the resulting more severe neuropathy. The lack of full protection might be explained by the slow kinetics of TPP, which would cause substantial NTE inhibition when PMSF effects on NTE had subsided. Since PMSF also affects the promotion site when given before initiation of neuropathy, the resulting neuropathy would then be due to both protection from and promotion of TPP effects by PMSF. No promotion by PMSF (120 mg/kg s.c.) was observed in TPP neuropathy (90 mg/kg i.v.) partially protected by PMSF (10–30 mg/kg s.c.) This might also be explained by the concurrent effects on NTE and on the promotion site obtained with PMSF pretreatment. We conclude that TPP neuropathy in the hen is likely to be the same as typical OPIDP. The unusual effects of combined treatment to hens with TPP and PMSF are explained by the prolonged pharmacokinetics of TPP and by the dual effect of PMSF i.e. protection from and promotion of OPIDP.

Keywords: Organophosphate delayed polyneuropathy; Hens; Neuropathy target esterase; Promotion; Protection; Triphenyl phosphite; Retrograde axonal transport

Effect of DMPS and various adsorbents on the arsenic excretion in guinea-pigs after injection with As₂O₃ by Franz-Xaver Reichl; Göran Hunder; Bernhard Liebl; Burckhard Fichtl; Wolfgang Forth (pp. 712-717).

The present experiments were performed to test the possibility of interrupting the enterohepatic circulation of arsenic (As). Therefore the efficacy of adsorbents to bind As and/or As-DMPS adducts in vitro and their effect on the excretion of As into the feces and urine in vivo were investigated after injection of As₂O₃ and DMPS in guinea-pigs. The adsorbents bentonite, activated charcoal or colestyramine, respectively, were tested. Only slight binding of ⁷³As (<5% of the ⁷³As dose) was observed to all adsorbents in vitro. After addition of DMPS, a good binding was found for ⁷³As to colestyramine (50%) or activated charcoal (60%), respectively. However, the ⁷³As-DMPS adduct was removed from the activated charcoal during washing. In the first in vivo experiment, male guinea-pigs (n=4/group) received As₂O₃ [0.02 mmol As(III)/kg s.c. labelled with a tracer dose of ⁷³As(III) (0.14 kBq/g)], 30 min later DMPS (0.1 mmol/kg i.p.) and by gastric tube (10 ml/kg body wt) either saline, bentonite (1 g/kg), activated charcoal (1 g/kg) or colestyramine (0.2 g/kg), respectively. Urine and feces were collected for 24 h. No increase in ⁷³As excretion into the feces was observed after administration of DMPS and all adsorbents, compared to control animals. In the second in vivo experiment male guineapigs (n=4/group) received the same As₂O₃ (+⁷³As)-and DMPS dose. In addition, with a gastric tube (10 ml/kg) saline, colestyramine (0.2 g/kg), DMPS (0.1 mmol/kg), or the combination of DMPS (0.1 mmol/kg) +colestyramine (0.2. g/kg) were administered according to the scheme given in the following table. The amount of feces excreted did not differ between groups. Excretion of ⁷³As within the feces during the first 12 h after As injection is shown in the following table (mean ±SEM). The same amount of ⁷³As (34 % of the ⁷³As dose) was excreted into the urine from animals in groups 4 and 5 during this time. Obviously, the combined oral administration of DMPS+cole-styramine markedly enhanced fecal excretion of As mobilized by DMPS i.p. It is suggested that interruption of enterohepatic circulation of As may be a valuable adjunct in the treatment of As poisoning.

Keywords: Arsenic binding in vitro; DMPS; Adsorbents; Bentonite; Activated charcoal; Colestyramine; Arsenic excretion; Feces; Urine; Guinea-pigs

Effect of lead on tube formation by cultured human vascular endothelial cells by Takuji Kishimoto; Tetsuhisa Oguri; Daizo Ueda; Manabu Tada (pp. 718-721).

The effect of lead acetate (Pb) on the growth of and tube formation by cultured human umbilical vascular endothelial cells (HUVEC) was examined. HUVEC were collected by enzymatic digestion with collagenase. The number of viable cells of HUVEC was negligibly affected by cultivation with Pb at concentrations of 1–100 μM, but was slightly reduced by cultivation at 500 μM. Tube formation was studied by culturing the cells on a gelled basement membranen matrix (Matrigel). Treatment of HUVEC with 0.1–50.0 μM Pb for 24 h inhibited tube formation dose-dependently. The length of tube formation decreased time-dependently with 1.0 μM Pb. These findings suggest that Pb inhibits the formation of a capillary network by HUVEC, and that Pb could be injurious to endothelials cell function.

Keywords: Endothelial cell; Lead; Tube formation; Basement membrane matrix

Inhibition of γ-glutamyltranspeptidase decreases renal deposition of mercury after mercury vapor exposure by C. -Y. Kim; C. Watanabe; Y. Kasanuma; H. Satoh (pp. 722-724).

The alteration of renal deposition of mercury (Hg) after mercury vapor (Hg^o) exposure was studied in mice pretreated with acivicin, a potent and irreversible inhibitor of γ-glutamyltranspeptidase (GGT). Pretreatment with acivicin decreased renal Hg concentration by about 60% and significantly increased Hg concentration in the urine compared with the non-treated group. The results suggest that renal deposition of Hg after Hg^o exposure depends on renal GGT, which plays an important role in the uptake of GSH or GSH conjugates filtered through the glomeruli. It seems that the mechanism of renal Hg deposition after Hg^o exposure is similar to that after exposure to ionic Hg: a GGT-mediated incorporation of an Hg-GSH complex into renal tubular cells. The acivicin pretreatment after Hg^o exposure did not affect Hg concentrations in the liver and erythrocytes.

Keywords: Mercury vapor (Hg^o); Renal deposition of mercury; γ-Glutamyltranspeptidase (GGT); Acivicin; Glutathione (GSH)

Hydrolysis rates of pyrrolizidine alkaloids derived from Senecio jacobaea by S. R. Dueker; M. W. Lamé; H. J. Segall (pp. 725-728).

Many of the commonly studied pyrrolizidine alkaloids (PAs) are built upon the subgroup retronecine (RET), which is released from the parent molecule by either base catalyzed or enzymatic hydrolysis of the ester linkages. The rate of appearance of RET in a hydrolytic study would thus reflect the rate of hydrolysis for the PA being tested. We have developed a gas chromatographic (GC) method to measure the release of RET from incubations of PAs with the guinea pig carboxylesterase, GPH1. The PAs tested were the following: jacobine (JAB), jacozine (JAZ), retrorsine (RES), and seneciphylline (SNP). The K_ms for SNP and JAZ were determined to be 64.9 and 349.2 μM, respectively. In addition, a qualitative assessment of hydrolytic activity toward a radiolabelled mixture of retrorsine/riddelliine (RES/RIL) was performed with HPLC and radiometric detection.

Keywords: Pyrrolizidine alkaloid; Seneciphylline; Jacozine; Senecio jacobaea ; Carboxylesterase; Guinea pig

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Archives of Toxicology (v.69, #10)