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Archives of Toxicology (v.69, #3)

A morphological analysis of the short-term effects of benzene on the development of the hematological cells in the bone marrow of mice and the effects of interleukin-1α on the process by Rodica Niculescu; George F. Kalf (pp. 141-148).

Chronic exposure of humans to benzene (BZ), a widely used industrial chemical and a ubiquitous environmental pollutant, causes aplastic anemia and acute myeloid leukemia. The purpose of the studies reported here was to determine whether the observed depression of bone marrow (BM) cellularity in mice administered benzene was reflected in a suppression of development of all of the hematopoietic lineages and to confirm the ability of interleukin-1α (IL-1α) to prevent BZ-induced BM cell depression. We report that BZ, administered twice per day for 2 days to C57Bl/6J mice at a dose of 600 mg/kg body weight, caused a significant depression of the total number of nucleated BM cells per femur when measured on day 3. The observed depression reflects a complex situation that represents the net effect of a decrease in the total number of cells of the lymphocytic and erythroid lineages, along with an increase in the number of intermediate and terminally differentiated cells of the granulocytic lineage. An experiment to monitor the effects of BZ over a 7-day period showed a progressive depressive effect on the lymphocytes and an initial depression of the erythroid cells at day 3 that remained constant until day 7. Conversely, the numbers of intermediate and terminally differentiated granulocytes progressively increased over the 7 days. The BM appeared to recover from the depressive effects of BZ immediately upon cessation of exposure, as the number of nucleated BM cells began to rise by day 5 and was equal to that of the control group by day 7. The results expand our earlier finding (Renz and Kalf 1991) that the overall depression of BM cellularity occurs because of an inability of the stromal fibroblast to produce colony-stimulating factors essential for stem and progenitor cell survival. This results from inhibition by the BZ metabolite, hydroquinone (HQ), of the processing of pre-IL-1α to the mature cytokine. The ability of exogenously administered biologically active, but not heat inactivated, recombinant IL-1α to prevent BZ-induced depression of total BM cellularity as a function of dose, when added concomitantly with BZ, and to augment the recovery from BZ exposure up to day 5 supports this hypothesis. IL-1α administered alone showed no effect on the total number of nucleated cells in the BM; however, analysis of its effects on the cells of the various lineages indicated that lymphocytes were unaffected, nucleated erythroid cell were decreased and the immature and mature granulocytic forms were increased. It is of interest that granulopoiesis is not decreased during BZ-induced inhibition of IL-1α production, but rather significantly stimulated.

Keywords: Benzene; Bone marrow depression; IL-1 Granulopoiesis

Characterization of the tobacco glycoprotein surface binding property of heart and skeletal muscle cells by Charles A. Santos-Buch; Harry R. Hall; Fausto Farfan; Irene Orlow; Adolfo Firpo; Betsy F. von Kreuter; Carl G. Becker (pp. 149-159).

Monolayers of L₆ rat skeletal myoblast cells formed surface binding isotherms with the purified tobacco leaf glycoprotein TGP₁ and the enriched cigarette tar glycoprotein TGP₂. Scatchard analysis showed that the binding in the range of the limited concentrations tested was to a single class molecule and the calculated affinity constant (K_d) for TGP₁ and TGP₂ showed similar values (9.78 × 10⁻¹³ M and 3.09 × 10⁻¹³ M, respectively). The bound TGPs were almost totally displaced by excess nonradiolabeled molecules. The calculated B_max of the L₆ myoblast monolayer was 2.93 fmol for TGP₁ and 0.217 fmol for TGP₂ per 32.2 mm². Guinea pig heart sarcolemma binding isotherms were also formed with radiolabeled TGP₁ and TGP₂. The interaction of tobacco leaf TGP₁ with the heart cell membranes was irreversible because only 15–20% of the bound TGP₁ was displaced by 100-fold, non-labeled molecules but the interaction of tar TGP₂ with heart sarcolemma was reversible and probably saturable. The heart sarcolemma TGP₂ affinity constant (K_d) was 5.88 × 10⁻⁷ M and the B_max, 2.45 × 10⁻⁸ M per 12.5 μg sarcolemma. Pretreatment of heart sarcolemma with increasing concentrations of leaf TGP₁ did not displace tar TGP₂ binding but its absorption on the membrane resulted in increased TGP₂ sarcolemma attachment by a complex and unexplained mechanisms. Increasing concentrations of the sera of 10 of 15 guinea pigs (67%) that received mainstream emissions of tobacco smoke from a University of Kentucky cigarette smoking machine for 152 days, displaced cigarette tar TGP₂ heart cell sarcolemma attachment and this inhibition was significantly different from that produced by the sera of sham smoked and of nonexposed animals (Mann-Whitney test, p=0.0082). Staphylococcus protein A inhibited the displacement of TGP₂ produced by the sera of cigarette smoke exposed guinea pigs and this observation indicated that this action was mediated by IgG molecules. The specific immunoprecipitation of a radiolabeled surface epitope of the L₆ myoblast monolayers pretreated with TGP₁ or TGP₂ by immune IgG against TGP₂ and by the IgG of an antiserum against standard TGP showed that the tobacco glycoproteins attached to a unit polypeptide of the plasma membrane of the muscle cells of approximately 76 kDa. These data support the notion that TGP molecules in cigarette smoke are absorbed systemically on smoking and may have a direct toxic effect when they attach to the surface TGP binding proteins of heart and skeletal muscle cells.

Keywords: Tobacco toxicity; Tobacco glycoprotein; Heart and skeletal muscle tobacco binding protein

Lectin binding pattern of schwann cells and macrophages in 2,5-hexanedione-induced axonal degeneration in rats by M. R. Carratù; M. Labate; S. De Santis; A. Giustino; M. A. De Salvia; V. Cuomo (pp. 160-164).

The lectin binding pattern of both Schwann cells and macrophages has been studied during axonal degeneration induced in the rat sciatic nerve by chronic administration of 2,5-hexanedione (0.8 ml/kg per day i.p. for 20 days). In particular, the present study aimed to establish a possible relationship between macrophage activation and expression of lectin binding sites. To identify and distinguish between Schwann cells and macrophages, electron microscopy was combined with the lectin staining method. On 2,5-hexanedione injury, a drastic disorganization of both axon and myelin sheath occurred and nerve fibers were replaced by a chain of ovoids. Besides the well-established concept that Schwann cells and macrophages cooperate in the removal of the myelin debris during axonal degeneration, evidence is presented that expression of binding sites to lectins is closely related to macrophage activation. Monocytes occasionally present in control nerves were labelled only by con A and sialidase-peanut sequence; in 2,5-hexanedione degeneration monocytes, prephagocytes (macrophages with minute bubbles) and phagocytes (macrophages with large bubbles) were labelled also by peanut, wheat germ and BSA I-B₄; moreover, phagocytes were labelled by soybean as well, thus showing a clearly differentiation-dependent binding pattern. Since changes in lectin binding pattern may reflect changes in complex carbohydrate structures, the results show that the expression of certain glycoproteins may be closely related to activation of macrophages in response to toxic injuries.

Keywords: Lectins; Macrophages; Axonal degeneration; 2,5-Hexanedione

Hepatotoxicity and P-4502E1-dependent metabolic oxidation of N,N-dimethylformamide in rats and mice by Elisabetta Chieli; Michela Saviozzi; Silvia Menicagli; Teresa Branca; Pier Giovanni Gervasi (pp. 165-170).

A comparative biochemical and histological study on the hepatotoxicity of a single dose of N,N-dimethylformamide (DMF) and N-methylformamide (NMF) in control and acetone-treated SD male rats and CD-1 male mice was performed. In control and acetone-pretreated rats, neither DMF nor NMF caused hepatic damage or elevation of plasma transaminases. In contrast, in acetonized but not in control mice, DMF administration yielded some evidence of liver necrosis and elevation of ALAT (alanine-amino transferase) activity. After a DMF dose of 1000 mg/kg, ALAT activity was found 1215 ± 832 mU/ml and 47 ± 18 mU/ml in acetonized and control mice, respectively. NMF treatment was hepatotoxic in control mice and lethal in acetonized mice. In control mice, an NMF dose of 600 mg/kg increased ALAT activity from a basal value of 35 ± 5 to 2210 ± 1898 mU/ml. When the oxidative metabolism of DMF was investigated, microsomes from both rats and mice preinduced by acetone increased the demethylation rate of DMF 7 to 10-fold compared to that (about 0.25 nmol/min per mg protein) of the corresponding control microsomes. The enzymatic affinities for DMF oxidation, however, were different: in mice the K_m (0.05 mM) was one order of magnitude lower than that (0.56 mM) found in rats. The experiments performed with purified rat and mouse P-450 2E1 in a reconstituted system confirmed that the P-450 2E1 isoforms are very active catalysts towards DMF oxidation (the turnover was about 10 nmol/min per nmol P-450 for both enzymes) but with a strikingly different affinity. Whereas the K_m for mouse P-450 2E1 was 0.08 ±0.03 mM, that for rat P-450 2E1 was 1.1 ± 0.2 mM. These findings indicate that the higher susceptibility of mice, compared with rats, to DMF hepatotoxicity, could be ascribed, at least in part, to the higher metabolic capacity of mouse P-450 2E1 with respect to that of rats.

Keywords: Dimethylformamide toxicity; Dimethylformamide metabolism; P-4502E1-dependent oxidation

Prostaglandin-H synthase mediated metabolism and mutagenic activation of 2-amino-3-methylimidazo [4,5-f] quinoline (IQ) by E. Wolz; D. Wild; G. H. Degen (pp. 171-179).

Prostaglandin-H synthase (PHS), a mammalian peroxidase of interest for the extrahepatic formation of reactive intermediates of carcinogens, catalyzes in vitro the metabolic activation of the mutagen and carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Incubation of ¹⁴C-labeled IQ with ram seminal vesicle microsomes (RSVM), a rich source of PHS, resulted in protein binding and generated products mutagenic in S. typhimurium YG1024. The mutagenic activity produced in IQ/PHS incubations was stable and extractable with ethyl acetate. Upon fractionation of such extracts by HPLC and subsequent analysis, two metabolites were identified as 2,2′-azo-bis-3-methylimidazo[4,5-f]quinoline (azo-IQ) and 3-methyl-2-nitro-imidazo[4,5-f]quinoline (nitro-IQ) confirmed by comparison of HPLC retention times, UV/VIS-, ¹H-NMR-spectroscopy, and mass spectrometry of synthesized standards. Azo-IQ was obtained by chemical oxidation of IQ with meta-sodium periodate. It was the major metabolite in PHS incubations, but has not been detected in monooxygenase incubations. Azo-IQ, without metabolic activation, was much less mutagenic in S. typhimurium YG1024 (308 rev/nmol) than nitro-IQ and 3-methyl-2-nitroso-imidazo[4,5-f]quinoline (nitroso-IQ), two other S9-independent mutagens which have been synthesized by chemical oxidation of IQ with sodium nitrite. Nitro-IQ was formed only in trace amounts but due to its potent mutagenicity in S. typhimurium YG1024 (2 × 10⁶ rev/nmol) it accounted for most of the mutagenic activity of the incubations. These data show that PHS-mediated in vitro metabolism of IQ results in its metabolic activation; thus PHS may contribute to the genotoxicity of IQ in extrahepatic tissues.

Keywords: 2-Amino-3-methylimidazo[4,5-f]quinoline Food mutagen; Prostaglandin-H synthase Metabolic activation

Dichloromethane as an inhibitor of cytochrome c oxidase in different tissues of rats by Andrea Lehnebach; Christian Kuhn; Dieter Pankow (pp. 180-184).

Based on the metabolism of dichloromethane (DCM) to carbon monoxide (CO), a process mediated by cytochrome P-4502E1 (CYP2E1), cytochrome c oxidase activity was determined in different tissues of rats after DCM exposure. It is likely that binding of CO to cytochrome c oxidase is significant at low carboxyhemoglobin levels, because intracellular effects of CO depend on CO partial pressures in the tissues. Two methods of exposure were used: (1) administration of DCM, 3.1, 6.2, and 12.4 mmol/kg p.o. in Oleum pedum tauri, 10% (v/v), producing a maximum of 10% COHb 6 h after gavage, and (2) accidental scenario, i.e. rats were exposed nose-only to DCM, 250 000 ppm for 20 s, producing 3–4% COHb after 2 h. Cytochrome c oxidase activity was reduced 6 h after the high oral DCM dose in brain, lung, and skeletal muscle by 28–42% and 20 min after inhalative uptake of DCM in the brain, liver, kidney, and skeletal muscle by 42–51%. COHb formation due to DCM, 6.2 mmol/kg p.o., was completely prevented after treatment of rats with the mechanism-based inhibitor of CYP2E1, diethyldithiocarbamate (DDTC), using an oral dose of 32 μmol/kg. The decrease in cytochrome c oxidase activity after exposure to DCM was not evident in rats treated with this dose of DDTC. Therefore, it seems that the effect of DCM is produced by the DCM metabolite CO.

Keywords: Dichloromethane; Metabolism; Carbon monoxide; Cytochrome P-4502E1; Diethyldithiocarbamate; Cytochrome c oxidase

Effect of cytochrome P450 isozyme induction and glutathione depletion on the metabolism of CS₂ to TTCA in rats by Helena Kivistö; Eivor Elovaara; Vesa Riihimaki; Antero Aitio (pp. 185-190).

Analysis of 2-thiothiazolidine-4-carboxylic acid (TTCA), a metabolite of carbon disulfide (CS₂), is used in the biological monitoring exposure to CS₂ at work. In order to clarify the metabolic reasons for individual variation in the urinary excretion of TTCA, the latter was studied in rats pretreated with model cytochrome P450 (CYP) enzyme inducers or glutathione (GSH) depletors. Ethanol, phenobarbital (PB) or 3-methylcholanthrene (MC) did not increase 24-h TTCA output following CS₂ inhalation (50 or 500 ppm, 6 h). After oral dosing (10 mg/rat), PB had an inhibiting effect on the excretion rate of TTCA. Tissue GSH depletors phorone, l-buthionine-RS)-sulfoximine (BSO) and diethylmaleate (DEM) decreased TTCA excretion in rats given an oral dose (10 mg/rat) of CS₂. The initial inhibition by phorone and DEM was reversed after 6 h and from 12 h onward the TTCA in urine exceeded the control level, an effect not seen with BSO. The proportion of CS₂ excreted in urine as TTCA within 24 h was 1.7% in control rats and 1% after BSO treatment, 1.3% after PB, 1.7% after acetone, 1.8% after MC, 2.0% after phorone and 2.5% after DEM treatment. The amount of TTCA in urine increased with the CS₂ dose in a nonlinear fashion: 1.6 μmol (50 ppm/6 h) vs. 4.9 μmol (500 ppm/6 h), and 0.2 μmol (1 mg/kg) versus 3.6 μmol (100 mg/kg). It is concluded that induction of different cytochrome P450 isoforms and transient glutathioen depletion have only minor effects on the disposition of TTCA in rats following low-level CS₂ exposure persistently low glutathione level as achieved by E.G. BSO, markedly decreased the metabolism of CS₂ to TTCA; these metabolic effectors are unlikely to have a major role in the individual variation of CS₂ metabolism in exposed workers.

Keywords: Carbon disulfide; TTCA (2-thiothiazolidine-4-carboxylic acid); Metabolism; Rats; Liver Glutathione; Cytochrome P450; Induction

Effects of inorganic mercury (Hg²⁺) on calcium channel currents and catecholamine release from bovine chromaffin cells by Frank Weinsberg; Ulf Bickmeyer; Herbert Wiegand (pp. 191-196).

The effects of inorganic mercury (Hg²⁺) on calcium channel currents and the potassium-evoked catecholamine release of bovine chromaffin cells in culture were examined. The effects of cadmium (Cd²⁺), known to block calcium channels and reduce catecholamine release of chromaffin cells, were studied for comparison. Calcium channel currents were recorded in the whole-cell configuration of the patch-clamp technique. Hg²⁺ is a potent calcium channel blocker in bovine chromaffin cells. The IC₅₀ value is about 3 μM, the Hill slope 1.46. In a concentration of 100 μM, Hg²⁺ blocked the currents completely; 100 μM Cd²⁺ had the same effect. Potassium-evoked catecholamine release from chromaffin cells was measured at different timepoints with high-performance-liquid-chromatography (HPLC) under control conditions and in the presence of different Hg²⁺ concentrations. Low Hg²⁺ concentrations (0.1 and 1 μM) did not affect the amount of the catecholamines epinephrine (E) and norepinephrine (NE) which was released. Under identical conditions 1 μM Cd²⁺ also had no effect on release. With 10 μM Hg²⁺ there was a time-dependent increase in the potassium-evoked catecholamine release (by 27% after 8 min). The E/NE ratio was not altered, suggesting that the release of both hormones was increased similarly. In contrast to this, the release was slightly reduced with 10 μM Cd²⁺. In the presence of 100 μM Hg²⁺, there was a reduction of the release during an early phase, followed by an increase. The reduction is most probably due to the fast and effective calcium channel block by Hg²⁺ in this high concentration. The calcium channel block by 100 μM Cd²⁺ also reduced the release significantly. Catecholamine release of bovine chromaffin cells is driven into two opposite directions by Hg²⁺. On the one hand, a calcium channel block reduces the release, while on the other hand effects occur which can increase the release. Both tendencies occur simultaneously, but have different concentration- and time-dependencies; therefore one can overcome the other under specific conditions. The catecholamine output at a given timepoint reflects the “sum” of these different effects.

Keywords: Inorganic mercury; Bovine chromaffin cells; Calcium channels; Catecholamine release; Cadmium

Stimulation of myosin light-chain kinase by Cd²⁺ and Pb²⁺ by Sheng Hao Chao; Chun Hui Bu; Wai Yiu Cheung (pp. 197-203).

The effect of Cd²⁺ on myosin light chain kinase (MLCK) reported in the literature is controversial, apparently because the level of Ca²⁺ contaminating the reaction mixture could not be accurately controlled by the addition of a metal chelator when Cd²⁺ was also present. In the present study, we have reduced the contaminating Ca²⁺ to a trace level that did not interfere with the enzyme activity; thus the use of a metal chelator was not necessary. We showed that Cd²⁺, or Pb²⁺ had a biphasic effect on MLCK isolated from chicken gizzard: stimulation at low and inhibition at high concentrations. (The stimulatory effect of on the enzyme activity isolated from chicken gizzard: stimulation at low and inhibition at high concentrations). The stimulatory effect of Cd²⁺ or Pb²⁺ on MLCK activity was not seen in the absence of calmodulin, and was abolished by trifluoperazine, a calmodulin antagonist, indicating that the heavy metals exert their activation via calmodulin. The inhibition of the enzyme activity by Cd²⁺ or Pb²⁺ at higher concentrations was also seen with the calmodulin-independent catalytic fragment of MLCK, suggesting that the inhibition is probably through their binding to sulfhydryl groups that are essential for catalytic activity. Pb²⁺ was more effective than Cd²⁺ in stimulating the enzyme activity, but less potent in inhibition. The extent of stimulation by heavy metals most likely resulted from a combination of the biphasic effects. Dithiothreitol and N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine selectively chelated Cd²⁺ and Pb²⁺ over Ca²⁺, and reversed their stimulatory or inhibitory effect on MLCK. Thus, they are useful agents to discriminate between the action of Ca²⁺ from that of these heavy metal cations. Cd²⁺ and Pb²⁺ not only activited MLCK in vitro, but also triggered smooth muscle contraction in an isolated rabbit aortic strip.

Keywords: Calmodulin; Cadmium; Lead; Heavy metal toxicity; Smooth muscle

Cytotoxicity of propyl gallate and related compounds in rat hepatocytes by Y. Nakagawa; S. Tayama (pp. 204-208).

The cytotoxic effects of propyl gallate (PG), its related gallates and gallic acid have been studied in freshly isolated rat hepatocytes. Addition of PG (0.5–2.0 mM) to hepatocyte suspension elicited concentration-dependent cell death accompanied by losses of intracellular ATP, adenine nucleotide pools, glutathione (GSH) and protein thiols. The rapid loss of intracellular ATP preceded the onset of cell death caused by PG. In the comparative toxic effects of PG and related gallates at concentration of 1 mM, octyl gallate (OG), dodecyl gallate (DG) and butyl gallate (BG) elicited an abrupt depletion of ATP, followed by an acute cell death. These gallates were more toxic than PG; the toxic effects of PG were similar to those of methyl gallate (MG) and ethyl gallate (EG). In mitochondria isolated from rat liver, PG caused a concentration-dependent increase in the rate of state 4 oxygen consumption, indicating an uncoupling effect. The rate of state 3 oxygen consumption was inhibited by OG and DG. According to the respiratory control index, the order of impairment potency to mitochondria was OG>BG, DG>PG>EG, MG>gallic acid. These results indicate that PG and related gallates are toxic to hepatocytes and that the acute cytotoxicity may be due to mitochondrial dysfunction.

Keywords: Propyl gallate; Rat; Hepatocytes; Cytotoxicity; Gallate esters; Antioxidant; Mitochondria

The detection of subchronic testicular damage using urinary creatine: Studies with 2-methoxyethanol by Michael Butterworth; Dianne Creasy; John A. Timbrell (pp. 209-211).

We have previously shown that a number of testicular toxicants administered acutely to rats raise urinary creatine. The aim of this study was to determine if this creatinuria was maintained during subchronic testicular damage. Repeated exposure of rats to 2-methoxyethanol for 10 days administered in the drinking water caused significant testicular damage at the highest dose. The urinary creatine: creatinine ratio was significantly increased in the animals receiving the highest dose (220 mg/kg per day) and also those receiving doses of 87 mg/kg per day. Increases in the ratio seen after the lowest dose (43 mg/kg per day) were significant in some cases, but showed more variability. Relative testicular weight was only significantly reduced after the highest dose. Increases in body weight over the time of exposure were only significantly lower after the highest dose of 2-methoxyethanol. The results indicate that urinary creatine may be a useful biomarker for chronic testicular damage.

Keywords: 2-Methoxyethanol; Testicular toxicity; Urinary marker; Creatine

Difference in rates of the reaction of various mammalian oxyhemoglobins with phenylhydrazine by Masahiro Kinuta; Jeanne L. Matteson; Harvey A. Itano (pp. 212-214).

Second order rate constants for the initial reaction of 12 mammalian oxyhemoglobins (Hb) with equimolar phenylhydrazine (PHZ), a compound inducing Heinz body hemolytic anemia, were determined by recording continuous changes in absorbance with time at 577 nm. The rate constants were varied in a range from 43 m⁻¹·s⁻¹ with pig Hb to 255 m⁻¹·s⁻¹ with dog Hb. On the other hand, isosbestic points at 526 and 587 nm were common to all the reaction processes. The aerobic reaction of Hb with PHZ resulted in denaturation of hemoprotein, and final reaction products were determined to be β-meso-phenylbiliverdin IXα and N-phenylprotoporphyrin IX. These results suggest that the reactivity of PHZ to Hb is influenced by the globin molecule, and the oxidative cleavage of the porphyrin ring causes the denaturation of hemoprotein.

Keywords: Phenylhydrazine; oxyhemoglobin; Phenylbiliverdin; Phenylprotoporphyrin; Bimolecular rate constant

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Archives of Toxicology (v.69, #3)