In vivo production of different chloroform metabolites: effect of phenobarbital and buthionine sulfoximine pretreatment.

The regioselective attack on microsomal phospholipid (PL) polar heads (PH) and fatty acyl chains (FC) demonstrated in vitro has been exploited for the selective quantitation in vivo of the biochemical damages produced by the oxidation and reduction products of CHCl3 metabolism. Five hours after CHCl3 injection (60 mg/kg body weight, ip) to control Sprague-Dawley rats, most of the label covalently bound in the liver was associated to PH, indicating a predominant production of COCl2. The levels of radioactivity bound to both PL moieties increased proportionally when 180 mg/kg body weight 14CHCl3 was administered. Buthionine sulfoximine (BSO) pretreatment resulted in a further increase of binding either to PH or FC. The pretreatment of rats with phenobarbital (PB) reduced the PH/FC binding ratio to 3.4, still indicating the predominance of the oxidative metabolism, but giving some indication of the simultaneous presence of CHCl3 reduction. When reduced glutathione (GSH) was depleted by BSO in PB-induced animals prior to 14CHCl3 administration, only the level of radioactivity associated with oxidative intermediates was increased six times. The present results confirmed that GSH is able to exert an efficient protection mainly toward 14CHCl3 oxidation intermediates. Furthermore, they indicate that in the liver of the Sprague-Dawley rat the major pathway of CHCl3 biotransformation is its oxidation and that pretreatment of rats with a GSH-depleting agent (such as BSO) is more relevant than PB induction in enhancing the biochemical damages produced by CHCl3.


Introduction
It is now widely accepted that chloroform can be both oxidized to phosgene (1,2) and reductively biotransformed in vitro and in vivo to dichloromethyl radical (3,4). Either type of reactive intermediates can react in vitro with cellular structures to form covalent adducts (5)(6)(7). Although the production of COCd2 has been associated with chloroform-induced acute toxicity (8)(9)(10), also the reductively produced metabolites can be involved in CHCl3 toxicity. In fact, CHC13 can stimulate lipid peroxidation in PB-pretreated Sprague-Dawley microsomal phospholipids rats (11)(12)(13), which are more susceptible to chloroform-induced hepatoxicity than control rats (14).
It has been shown in vitro that PL are the major site of CHC13-induced damage, in experimental conditions resembling the physiological status of the liver (6,7). Moreover, our recent results, obtained in vitro by means of chemical transmethylation or of enzymatic hydrolysis with phospholipase C (15), indicated that the different reactive intermediates (namely, COCd2 and .CHCl2) show a typical regioselectivity in the attack to PL. Indeed, while COCd2 binds preferentially to PL polar heads, PL fatty acyl chains are the main target for dichloromethyl radicals.
This feature has been exploited in this preliminary study for the selective in vivo quantitation of the binding due to each type of intermediates to assess if the two metabolic pathways are simultaneously active in the liver of Sprague-Dawley rats.
Furthermore, we investigated different effects of PB, a well-known enhancer of CHCl3 toxicity (16,17), and of BSO, which is able to deplete GSH in vivo (18), on the two metabolic pathways of CHCl3.

Aninals
Male Sprague-Dawley rats (180-200 g) were from Nossan (Italy). They were maintained on a 12-hr light cycle and provided food and water ad libitum for 1 week before the start of the experiments.
When required, animals were pretreated with 0.1% (w/v) PB sodium salt in drinking water for 1 week; BSO, dissolved in distilled H20 was injected ( Lipoluma. Data were compared by means of the Student's t-test.

Results
After the ip administration of 60 mg/kg body weight 14CHCI3 to control male Sprague Dawley rats, most of the label covalently bound in the liver was associated to PL hydrophilic PH (Figure 1). Indeed the binding was about 6-fold higher than the label bound to FC. This amount of radioactivity bound either to PH or FC increased proportionally when a 3-fold higher dose (180 mg/kg body weight) was administered ( Figure 1).
When control rats were pretreated with BSO prior to 14CHCI3 administration, the effect of GSH depletion on covalent binding could be observed. The total amount of radioactivity was 7-fold higher (Figure 2). The binding associated to both microsomal PL moieties resulted similarly affected (no significant changes in the ratio PH/FCassociated label). The pretreatment of rats with PB nearly doubled the total PL covalent binding due to 14CHC13 metabolites (Figure 3).
The radioactivity bound to FC increased slightly but significantly more than that one associated to PH.
When BSO was administered to PBinduced animals prior to 14CHC13 injection, the level of total radioactivity bound to PL was 6.5 fold increased with respect to that measured in PB-induced rat liver ( Figure 3). The variation was due only to the increase of radioactivity associated to PH, since the level of covalent binding to FC was not significantly changed.

Discussion
Injection of a single subtoxic dose of 14CHCl3 to variously pretreated Sprague-Dawley rats resulted in a predominant binding of the 14C label to the PH of hepatic microsomal PL, indicating that COC12 is the major product of CHCl3 bioactivation. Indeed, the ratio of radioactivity associated with PH with respect to that of FC (PH/FC) was about six and remained almost constant at the two dosage levels tested of 14CHCI3. The observed binding to FC may not be attributed with certainty to the radicals derived from CHCI3 reduction, since the regioselectivity of binding to PL shown by the two CHCl3 metabolites is not absolute (15). This finding is in line with previous in vitro results (7,15), suggesting that in hepatic microsomes from control Sprague-Dawley rats in experimental conditions mimicking the physiologic oxygenation of the liver, reductive metabolism is not significantly expressed or requires very low P02 values. Pretreatment of Sprague-Dawley rats with PB prior to 14CHCl3 caused the binding to FC to increase significantly more than the binding to PH. The PH/FC binding ratio decreased to 3.4.
Environmental Health Perspectives These data indicate the predominance of CHC13 oxidative metabolism, and suggest that some contribution by the reductive pathway is also present.
The marked increase of total PL binding (together with the increase of PH/FC binding ratio up to 21.8) in liver microsomes of rats pretreated with BSO indicated that GSH exerts in vivo a highly efficient scavenging action toward the CHC13 intermediate produced through the oxidative pathway. The greater susceptibility to CHC13-induced hepatotoxicity observed in GSH-depleted rats (16) may be associated mainly to a decreased protection against COC12, the major product of CHCI3 oxidation (1,2,8).
These preliminary results suggest that in the liver of either control or PB-induced Sprague-Dawley rats the predominant biotransformation pathway of CHC13 is its oxidation and that the damages exerted by CHC13 on the microsomal PL are potentiated by GSH-depleting agents such as BSO, more than PB-like inducers of CHC13 metabolism.