Effect of long-term ethanol pretreatment on the metabolism of dichloromethane to carbon monoxide in rats.

The present study investigates the influence of long-term ethanol (ETOH) treatment of rats [10% (v/v) for 4, 12, and 36 weeks] on the metabolism of DCM after its oral and inhalative uptake to CO. Biotransformation of DCM to CO as measured by carboxyhemoglobin (COHb) formation was stimulated after long-term ETOH treatment in rats. A single oral dose of DCM (6.2 mmol/kg body mass) caused a significant increase of COHb, the maximum of about 9% occurring approximately 6 hr after DCM administration. In comparison to this control, in the blood of rats pretreated with ETOH (10% v/v) for 4, 12, and 36 weeks COHb values of 18, 17, and 13%, respectively, were measured. Long-term ETOH treatment followed by inhalation of 100, 500, and 2500 ppm DCM for 4 hr stimulated the formation of COHb, compared to controls. The elevation of COHb level was accompanied by decreased concentrations of DCM in the blood. The reason for the elevated biotransformation of DCM was ascertained by means of the determination of p-nitrophenol and aniline hydroxylation in liver microsomes of rats after long-term ETOH treatment to be an increase in cytochrome P450-dependent enzyme activities.

Chlorodibromomethane metabolism to bromide and carbon monoxide in rats.

The chlorodibromomethane (CDBM) metabolites bromide and CO were analysed as bromide level in plasma and carboxyhaemoglobin (COHb) level in blood of rats, respectively. The mean basic levels of bromide in plasma of rats receiving vehicle were 0.075 +/- 0.036 mmol/l (n = 27). After administration of CDBM at 0.4, 0.8, 1.6, and 3.1 mmol/kg p.o., the mean bromide levels rose to maximal values that were higher by a factor 27, 48, 69, and 135, respectively. Bromide elimination was slow and the plasma level was significantly increased following repeated administration in comparison to a single administration of CDBM. The CDBM concentrations in blood and in fat tissue 6 h after the last of 7 administrations of 0.8 mmol CDBM/ kg p.o., once a day for 7 consecutive days, were significantly lower than 6 h after a single gavage of this CDBM dose. The mean normal level of 0.45 +/- 0.32% COHb in rats (n = 30) was significantly increased following oral CDBM uptake. Initially higher COHb levels were measured after 7 consecutive applications of 0.8 mmol/kg CDBM. After a single administration of CDBM the level of glutathione disulphide in the liver was significantly increased; this effect was reversible. The oxidative CDBM metabolism was influenced by the glutathione (GSH) concentration in the liver. The rate of COHb and bromide formation was decreased after GSH depletion due to pretreatment of rats with buthionine sulphoximine (BSO) and increased following enhancement of the GSH concentration due to pretreatment of the animals with butylated hydroxyanisole (BHA). CDBM is a substrate for cytochrome P-450 2E1 (CYP2E1), as demonstrated by the inhibition of bromide and COHb formation due to simultaneous administration of CDBM and the CYP2E1 inhibitor diethyldithiocarbamate (DDTC); also by the initially higher levels of bromide in plasma and COHb in blood after gavage of CDBM pretreated with isoniazid (INH), an inducer of CYP2E1. The increase of bromide formation after CDBM administration in phenobarbital (PB)-pretreated rats indicated that cytochrome P-450 2B1 and 2B2 (CYP2B1 and CYP2B2) play a role as catalysts of the CDBM biotransformation. It is shown that m-xylene pretreatment, which activates CYP2E1 as well as CYP2Bs, leads to a higher bromide level after CDBM administration than the INH or PB pretreatment. In liver microsomes of rats treated with CDBM (0.8 mmol/kg p.o., seven daily applications), the p-nitrophenol hydroxylase (p-NPH) activity, a market of CYP2E1, was increased. It is concluded that CDBM may be an inducer of CYP2E1. These results combined with literature data demonstrate that the oxidation of CDBM was catalysed mainly by CYP2E1 and CYP2Bs and that there may be a risk of bromide accumulation following repeated uptake of the trihalomethane.

Cardiotoxicity of chlorodibromomethane and trichloromethane in rats and isolated rat cardiac myocytes.

The cardiovascular effects were investigated after acute and subacute treatment with chlorodibromomethane (CDBM; 0.4 to 3.2 mmol/kg p.o.), trichloromethane (TCM; 0.31 and 1.25 mmol/kg p.o.) and mixtures of CDBM and TCM (acute, 0.8 mmol CDBM/kg + 1.25 mmol TCM/kg p.o.; subacute, 0.4 mmol CDBM/kg + 0.31 mmol TCM/kg p.o.) in conscious and urethane anaesthetized male Wistar rats (n = 6-10 per treatment). Furthermore it was observed whether cardiovascular responses were modified in CDBM or TCM treated rats after administration of exogenous catecholamines (epinephrine, 1 microg/kg; norepinephrine, 2 microg/kg) and underpinned with in vitro alterations of Ca2+ dynamics in cardiac myocytes. The present findings demonstrated that single and subacute oral administration of CDBM or TCM and mixtures of CDBM and TCM resulted in arrhythmogenic and negative chronotropic and dromotropic effects in conscious and urethane anaesthetized rats. The atrioventricular conduction time and the intraventricular extension time were extended. A slight shortening of the repolarization velocity was observed. The myocardial contractility was depressed and the heart was sensitized to the arrhythmogenic effects of epinephrine. After catecholamine injection the adrenergic cardiovascular responses in urethane anesthetized rats were modified: increased hypertensive epinephrine and norepinephrine action as well as augmentation of negative chronotropic and negative dromotropic cardiac effects of catecholamines were observed. The positive inotropic adrenergic response was diminished. The present in vivo findings, myocardial depression after acute CDBM treatment, as determined by different indices of contractility, correlate well with the observed inhibitory actions of CDBM on Ca2+ dynamics in isolated cardiac myocytes. All cardiovascular alterations found after CDBM or TCM treatment were not intensified after treatment with mixtures of CDBM and TCM. The effects observed were distinctly stronger after TCM (1.25 and 0.31 mmol/kg) treatment compared to CDBM (0.8 and 0.4 mmol/kg) treatment.

Induction of hepatic cytochrome P4502E1 in rats by acetylsalicylic acid or sodium salicylate.

Studies were conducted on the mechanism of the ethanol-inducible cytochrome P450 (cytochrome P4502E1, CYP 2E1) induction by acetylsalicylic acid (ASA) or its metabolite salicylate (SAL). Many exogenous inducers of CYP 2E1 seem to increase CYP 2E1 by post-transcriptional activation without elevation of its mRNA level. Administration of a single high dose of ASA or SAL produces a significant increase in the activity of the hepatic microsomal p-nitrophenol hydroxylase in rats. Pretreatment of ASA-treated rats with a blocker of mRNA transcription, actinomycin D, or a blocker of protein synthesis, cycloheximide, markedly suppressed this enhanced activity of microsomal p-nitrophenol hydroxylase. The CYP 2E1 mRNA levels in livers of control rats and rats treated with ASA or SAL were measured by Northern blot analysis. Significantly elevated CYP 2E1 mRNA levels were measured in livers of treated rats compared with mRNA amounts of the control group. These data suggest that mRNA elevation seems to be characteristic for ASA induction, while other inducing agents show different patterns and mechanisms of activation.

Dichloromethane as an inhibitor of cytochrome c oxidase in different tissues of rats.

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, diethyl-dithiocarbamate (DDTC), using an oral dose of 32 mumol/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.

Acetylsalicylic acid--inducer of cytochrome P-450 2E1?

Pretreatment of rats with acetylsalicylic acid or sodium salicylate stimulates the metabolism of dichloromethane to carbon monoxide as measured by the carboxyhemoglobin level in blood. Simultaneous administration of dichloromethane and acetylsalicylic acid or sodium salicylate, respectively, was accompanied by reduced carboxyhemoglobin formation. In liver microsomes of rats pretreated with acetylsalicylic acid the p-nitrophenol hydroxylase activity was increased. It is concluded that (i) cytochrome P-450 2E1 is involved in the metabolic conversion of both dichloromethane and salicylic acid, and (ii) salicylic acid may be an inducer of cytochrome P-450 2E1.

Effect of methanol or modifications of the hepatic glutathione concentration on the metabolism of dichloromethane to carbon monoxide in rats.

1. The metabolism of dichloromethane (DCM) to carbon monoxide as measured by the carboxyhaemoglobin (COHb) level in the blood is stimulated by pretreatment with methanol (MET). After simultaneous administration of both DCM, 6.2 mmol kg-1 p.o., and MET, > 148 mmol kg-1 p.o., the COHb formation is inhibited. 2. MET ingestion results in a transient decrease of the glutathione (GSH) content of the liver. In rats treated with GSH-depleting chemicals such as diethylmaleate (DEM), phorone (PHO), or buthionine sulphoximine (BSO) there were no enhancements of the carboxyhaemoglobinaemia caused by DCM. The COHb formation was not influenced by an increase of the hepatic GSH concentration due to repeated administration of butylated hydroxyanisole (BHA). 3. It is concluded that cytochrome P450 IIE1 (CYP 2E1) is responsible for the metabolic interaction of both DCM and MET, and MET may be an inducer of CYP 2E1. The two pathways of DCM, the oxidative via CYP 2E1 and the metabolism via GSH/GSH-S-transferase seem to be independent.

Effect of isoniazid or phenobarbital pretreatment on the metabolism of dihalomethanes to carbon monoxide.

An oral dose of 6.2 mmoles of diachloromethane (DCM), bromochloromethane (BCM) or dibromomethane (DBM) per kg body mass yielded a maximum carboxyhemoglobin (COHb) level of about 9% (at 6 hr), 11% (at 8 hr) and 22% (at 12 hr), respectively. Pretreatment of rats with isoniazid, 4 x 0.36 mmol/kg i.p., produced significant enhancements of the COHb formation; the values were 18.0 +/- 0.8% COHb after DCM, 24.1 +/- 0.8% COHb after BCM, and 39.0 +/- 1.3% COHb after DBM. Prior administration of phenobarbital, 4 x 0.31 mmol/kg i.p., caused no appreciable alterations in the COHb levels after DCM and slight but significant increases after BCM as well as after DBM. The data indicate that the oxidative metabolism of dihalomethanes to carbon monoxide is mainly catalyzed by cytochrome P-450 IIE1 and that the DCM-evoked COHb formation seems to be a method of testing whether a chemical is an inducer of this form of cytochrome P-450 in vivo.

Influence of aromatic hydrocarbons on the metabolism of dichloromethane to carbon monoxide in rats.

The influence of prior or simultaneous oral administration of benzene, toluene, o-, m-, or p-xylene on the carboxyhemoglobin (COHb) level after a single dose of dichloromethane (DCM) was investigated in male rats. Six hours after administration of DCM, 6.2 mmol/kg, the mean maximum COHb level was 9.3 +/- 1.9%. This level was significantly enhanced by prior administration of benzene (16.9 mmol/kg) at 12-24 h, of toluene (18.8 mmol/kg) at 20-28 h, of o- (16.6 mmol/kg) and m-xylene (16.3 mmol/kg) at 20-32 h, and of p-xylene (16.2 mmol/kg) at 24-32 h. The corresponding maximum COHb levels were 20.7 +/- 1.3, 18.6 +/- 1.1, 18.9 +/- 1.1, 22.7 +/- 1.2, and 13.2 +/- 1.0%, respectively. After simultaneous administration of both DCM and the aromatic solvent, the COHb formation was inhibited: values of 1.3 +/- 0.3, 1.7 +/- 0.4, 3.6 +/- 0.2, 1.9 +/- 0.2, and 2.0 +/- 0.2% COHb, respectively, were found. The inhibition was also evident when DCM was administered 12 h after toluene or m-xylene and 12, 16 or 20 h after p-xylene. The inhibition was dose-related as seen after combined gavage of o-, m-, or p-xylene and DCM. The o- and m-, but not the p-methylhippuric acid (MHA) excretion in the urine was significantly reduced after simultaneous administration of equimolar doses of DCM and the corresponding xylenes. In conclusion, it seems that the stimulation or inhibition of the COHb formation after DCM caused by pretreatment with or by simultaneous administration of the aromatic solvents is due to the induction of cytochrome P-450 IIE1 or to competition between DCM and the aromatic solvent on this isozyme of cytochrome P-450.

[Effect of carbon tetrachloride and chloroform on hematologic parameters in rats]. / Einfluss von Tetrachlorkohlenstoff und Chloroform auf hämatologische Parameter bei Ratten.

While the hepato- and nephrotoxicity of carbon tetrachloride (CCl4) in men and animals are well characterized, little is known about the haematological effects of CCl4 poisoning. Therefore some haematological parameters were investigated after oral administration of CCl4 and the CCl4-metabolite chloroform in rats. The haemoglobin concentration in the blood was significantly reduced 7 days after administration of CCl4. Chloroform produced a decrease of the relative reticulocyte count at the same time. All effects are moderate and reversible.

Enhancement of dichloromethane-induced carboxyhemoglobinemia by isoniazid pretreatment.

Pretreatment of rats with isoniazid (8 x 0.36 mmol/kg i.p. or 1 x 0.36 mmol/kg i.p.) produced dichloromethane-induced carboxyhemoglobin concentrations in the blood which are 2.6 and 1.8 times higher than carboxyhemoglobinemia after administration of dichloromethane per se. Simultaneous administration of both compounds reduced the dichloromethane metabolism to carbon monoxide as measured by carboxyhemoglobin level in the blood.

Interaction of dichloromethane and ethanol in rats: toxicokinetics and nerve conduction velocity.

The influence of a single combined administration of ethanol (174 mmol/kg per os) plus dichloromethane (1.6, 6.2, or 15.6 mmol/kg p.o.) on blood concentrations of the tested substances and of carboxyhemoglobin, and on nerve conduction velocity was studied in rats. The blood alcohol concentration was not influenced significantly by dichloromethane. The single high dose of ethanol completely inhibited the carboxyhemoglobin concentration increase due to dichloromethane, but did not prevent the dichloromethane-induced decrease of nerve conduction velocity. It produced initially lower, then higher concentrations of dichloromethane in blood than values seen after administration of dichloromethane per se. Rats treated with ethanol plus dichloromethane showed a more pronounced decrease of nerve conduction velocity compared with rats administered dichloromethane only.

Effect of single dichloromethane administration on the adrenal medulla of male albino rats.

Adult male albino rats received a single dose of dichloromethane (DCM) orally. In response to administrations of DCM, 6.2 or 9.4 mmol/kg body mass, urinary catecholamine excretion increased significantly. 4 h and 48 h following a single DCM dose of 15.6 mmol/kg body mass, morphological investigations revealed cytological changes and a distinct reduction of chromaffin reaction in the adrenal medulla. The norepinephrine contents of cells were decreased strongly 4 h after DCM administration and increased weakly again, but distinct reduced in comparison with the control animals 48 h later. The mean nuclear volumes in the norepinephrine and epinephrine cells were enhanced significantly 4 h as well as 48 h after acute DCM intoxication.

[Effect of chronic alcohol administration on the adrenals thyroid, pancreatic islands and liver of male albino rats]. / Wirkung chronischer Ethanolapplikationen auf die Nebennieren, die Schilddrüse, das Inselorgan und die Leber männlicher Albinoratten.

In response to chronic oral ethanol administration, the adrenals, thyroid glands, pancreatic islets, and livers of male white rats show histological and chemomorphological reactions. Cytological, karyometrical, and histochemical findings from adrenal medulla indicate increased secretion and synthesis of catecholamines. Adrenal cortex shows morphokinetic changes within the meaning of progressive transformation. About 1 h after the last ethanol administration, there are no signs of inappropriate insulin secretion in the B-cells of pancreatic islets. In addition to a small lipid storage and a marked decrease of glycogen contents, the hepatocytes show karyometrical sign of an increased cell metabolism.