4,4'-Methylenebis(2-chloroaniline) (MOCA): The Effect of Multiple Oral Administration, Route, and Phenobarbital Induction on Macromolecular Adduct Formation in the Rat

The effect of multiple oral administration of MOCA, a suspecthuman carcinogen, was studied in the adult male rat. As manyas 28 consecutive daily doses of [¹⁴C]MOCA at 28.1µmol/kgbody wt (5 µC1/day) were administered and rats were euthanizedat weekly intervals for 7 weeks. MOCA adduct formation for globinand serum albumin was evaluated by determination of [¹⁴C]MOCAcovalent binding. The covalent binding associated with globinshowed a linear increase over the 28-day exposure period with342 fmol/mg globin 24 hr after the final dose. More extensivecovalent binding was detected for albumin with 443 fmol/mg albuminafter the final dose, but increases were not linear. After cessationof dosing, the albumin adduct levels decreased rapidly (t _1/2=4.6 days) in relation to globin adduct levels (t _1/2 =16.1days). The MOCA-globin adduct t _1/2 is consistent with thatdetermined after a single 281 µmol/kg oral dose of MOCA.Significant differences related to route of administration weredetected for 24-hr globin covalent binding with ip > po >dermal. Distribution of undifferentiated [¹⁴C]MOCA was highestin the liver at 24 hr with tissue levels for liver > kidney> lung > spleen > testes > urinary bladder. Inductionof cytochrome P450 enzymes by administration of phenobarbital(100 mg/kg/day/3 days) resulted in a significant (p < 0.05)increase in MOCA-globin adduct formation detected with 33.5pmol/ mg globin for induced rats versus 13.6 pmol/mg globinfor control rats. Although MOCA-globin and albumin adducts showdiffering stability, quantification of such MOCA adducts maybe useful for long-term industrial biomonitoring of MOCA.     

Biological Markers of Acute Acrylonitrile Intoxication in Rats as a Function of Dose and Time

Three markers of acute acrylonitrile (AN) intoxication, namely,tissue glutathione (GSH), tissue cyanide (CN), and covalentbinding to tissue protein, were studied as a function of doseand time. Doses administered and responses expected were 20mg/kg (LD0), 50 mg/kg (LD10), 80 mg/kg (LD50), and 115 mg/kg(LD90). Liver GSH was the most sensitive marker of AN exposure.At 80 mg/kg AN, virtually complete depletion of liver GSH wasobserved within 30 min with no recovery through 120 mm. KidneyGSH showed a similar, but less intense depletion; while bloodand brain GSH were more refractory to AN. Whole blood and brainCN rose progressively during the first 60 mm in a dose-dependentfashion. At the lowest dose, CN levels decreased thereafter,whereas, at the three higher doses, CN levels were maintainedor continued to increase through 120 min. At the highest dose,blood and brain CN remained at acutely toxic levels through240 mm. Covalent binding increased rapidly in all tissues duringthe first 30 mm at all doses. At the lowest dose, little additionalcovalent binding was observed beyond 30 mm, while at the threehigher doses, covalent binding increased, although at a slowerrate. The data indicate that these three biologic markers ofacute AN intoxication respond dramatically in a time-dependentmanner in the toxic dosage range. Furthermore, the data provideevidence that AN toxicity is gated by GSH depletion in liverwith the resultant termination of AN detoxification.     

4,4'-Methylene-bis(2-chloroaniline) (MOCA): Comparison of Macromolecular Adduct Formation after Oral or Dermal Administration in the Rat

4,4'-Methylene-bis(2-chloroaniline) (MOCA): Comparison of MacromolecularAdduct Formation after Oral or Dermal Administration in theRat. CHEEVER, K. L., RICHARDS, D. E., WEIGEL, W. W., BEGLEY,K. B., DEBORD, D. G., SWEARENGIN, T. F., AND SAVAGE, R. E. JR.(1990). Fundam. Appl. Toxicol. 14, 273–283. The macromolecularbinding of 4,4'-methylene-bis(2-chloroaniline) (MOCA), a suspecthuman carcinogen, was studied in the adult male Sprague-Dawleyrat after both oral and dermal administration. Rats were euthanized1, 3, 7, 10, 14, and 29 days after a single 281 µmol/kgbody wt dose of [¹⁴C]MOCA (oral, 213 µCi/kg; dermal, 904µCi/kg). DNA from various tissues and hemoglobin wereisolated for determination of the time course of MOCA macromolecularbinding. After oral administration adduct formation was rapidwith maximum levels appearing at 24 hr. The 24-hr covalent bindingassociated with the globin was 7.84 pmol/mgglobin (t? = 14.3days). More extensive 24-hr covalent binding was detected forliver DNA with 49.11 pmol/mg DNA (t? = 11.1 days). After dermaladministration of MOCA the major portion of the dose, 86.2%,remained at the application site throughout the study. For theserats the 24-hr covalent binding determined for liver DNA was0.38 pmol/mg DNA (t? = 15.6 days). Although lower levels weredetected after dermal application, similar stability of MOCA-DNAadducts indicates that quantification of such MOCA adducls maybe useful for the long-term industrial biomonitoring of MOCAexposure and for the evaluation of human DNA-MOCA adduct formation,a lesion thought to be associated with the production of cancer.     

Temporal and Dose-Response Features of Monochlorobenzene HepatotoxicityBN in Rats

Temporal and Dose–Response Features of MonochlorobenzeneHepatotoxicity in Rats. DALICH, G. M., AND LARSON, R. E. (1985).Fundam. Appl. Toxicol. 5, 105–116. Time- and dose-dependentcorrelations of monochlorobenzene (CB) hepatotoxic effects werestudied in view of (1) assumed mechanistic similarities to bromobenzene(BB), (2) the paucity of these data for CB, and (3) the relativelygreater environmental importance of CB compared with BB. Anip dosage of 9.8 mmol/kg CB (LD10) produced evidence of livertoxicity over a 72-hr time course. Sulfobromophthalein (BSP)retention was maximized 3–16 hr post-treatment and normalizedafter 72 hr, whereas plasma alanine aminotransferase activity(ALT) and morphological evidence of damage were maximized about48 hr after dosing. Maximal covalent binding to liver protein(3.07 nmol/mg) had occurred by 24 hr and approximately 36% ofthe administered dose had appeared in the urine by 48 hr. Liverand plasma CB concentrations were proportionally increased overthe dosage range 2.0–14.7 mmol/kg but marked centrolobularnecrosis and ALT elevations were seen only at the two highestdosages (9.8 and 14.7 mmol/ kg). On the other hand, all dosesdepressed hepatic glutathione (GSH) to between 30 and 40% ofcontrol by 4 hr. Evidence of rapid recovery was evident at 2.0and 4.9 mmol/kg but GSH levels remained low through 8 hr after9.8 or 14.7 mmol/kg. Liver/body weight ratios were increasedto a similar extent at all dosages when measured 24 hr post-treatment.Urinary excretion ranged from 59% at the low dosage to only19% at the highest dosage by 24 hr. Dose-related covalent bindingto liver protein at 24 hr occurred up to 9.8 mmol/kg but thebinding associated with 14.7 mmol/kg was equivalent to thatseen with the 4.9 mmol/kg dosage (1.6 nmol/mg protein). CytochromeP-450 levels were depressed to between 50 and 80% of control24 hr post-treatment with no clear dose relationship. Whilethe hepatotoxic effects of CB and BB appear similar, these datasuggest that some mechanistic differences are involved.     

Metabolic activation of the antidepressant tianeptine. II. In vivo covalent binding and toxicological studies at sublethal doses

Administration of [14C]tianeptine (0.5 mmol/kg i.p.) to non-pretreated hamsters resulted in the in vivo covalent binding of [14C]tianeptine metabolites to liver, lung and kidney proteins; this very high dose (360-fold the human therapeutic dose) depleted hepatic glutathione by 60%, and increased SGPT activity 5-fold. Lower doses (0.25 and 0.125 mmol/kg) depleted hepatic glutathione to a lesser extent and did not increase SGPT activity. Pretreatment of hamsters with piperonyl butoxide decreased in vivo covalent binding to liver proteins, and prevented the increase in SGPT activity after administration of tianeptine (0.5 mmol/kg i.p.). In contrast, pretreatment of hamsters with dexamethasone increased in vivo covalent binding to liver proteins, and increased SGPT activity after administration of tianeptine (0.5 mmol/kg i.p.). Nevertheless, liver cell necrosis was histologically absent 24 hr after the administration of tianeptine (0.5 mmol/kg i.p.) to non-pretreated or dexamethasone-pretreated hamsters. In vivo covalent binding to liver proteins also occurred in mice and rats, being increased by 100% in dexamethasone-pretreated animals. In vivo covalent binding to liver proteins was similar in untreated female Dark Agouti rats and in female Sprague-Dawley rats. These results show that tianeptine is transformed in vivo by cytochrome P-450, including glucocorticoid-inducible isoenzymes, into chemically reactive metabolites that covalently bind to tissue proteins. The metabolites, however, exhibit no direct hepatotoxic potential in hamsters below the sublethal dose of 0.5 mmol/kg i.p. The predictive value of this study regarding possible idiosyncratic and immunoallergic reactions in humans remains unknown.     

2-Methylfuran toxicity in rats--role of metabolic activation in vivo

Administration of a single ip dose of 2-methylfuran (2-MF) to male Sprague-Dawley rats at a dose of 100 mg/kg produced centrilobular necrosis of the liver and bronchial injury of the lung, the severity of the lesions increasing with increasing doses up to 400 mg/kg. Kidneys, however, showed no visible evidence of tissue damage even at the highest dose. Liver injury was also evidenced by an increase in serum glutamic pyruvic transaminase (SGPT) levels. Tissue distribution and covalent binding studies conducted over a dose of 50-200 mg/kg of [14C]2-MF indicated that the total radioactivity present per gram of wet tissue was in the order of liver greater than kidney greater than lung greater than blood. Covalent binding of the label to protein was greatest in the liver followed by kidney and the lung. Radioactivity bound covalently per milligram of DNA was also highest in the liver followed by kidney. Tissue distribution and covalent binding studies were conducted over a period of 0.5 to 24 hr after an ip dose of 100 mg/kg of [14C]2-MF. Maximal covalent binding was observed in the liver at 4 hr. At all time points binding of the label was greatest in liver, followed by kidney. Liver glutathione levels were depressed following 2-MF administration. Pretreatment of rats with phenobarbital markedly increased the covalent binding to protein and DNA and caused a twofold increase in SGPT compared to rats treated with 2-MF alone. Pretreatment with 3-methylcholanthrene had no effect on either parameter. Administration of N-octylimidazole, an inhibitor of cytochrome P-450, prior to administration of the radiolabeled 2-MF decreased the covalent binding of the label to protein and DNA. Moreover, the SGPT levels remained the same in the pretreated rats compared to the rats treated with vehicle alone. Thus, pretreatment with phenobarbital, an inducer of cytochrome P-450, enhanced both covalent binding and toxicity while prior treatment with N-octylimidazole, an inhibitor of cytochrome P-450 decreased covalent binding and prevented hepatotoxicity of 2-MF. These results support the view that at least some of the toxic effects of 2-MF are mediated by reactive metabolite(s) formed in vivo.     

Effects of glutathione-modulating agents on the covalent binding and toxicity of dichlobenil in the mouse olfactory mucosa.

Twenty-four hours following injection of a single dose of the herbicide dichlobenil (2,6-dichlorobenzonitrile) in C57Bl/6 mice a steep dose-response curve for the histopathological toxicity in the olfactory mucosa was observed. Four hours following injection of a toxic dose of [ring-14C]dichlobenil (12 mg/kg) the covalent binding in the olfactory mucosa was 26 times higher than that in the liver. A dose-dependent decrease of nonprotein sulfhydryls (mainly glutathione, GSH) in the olfactory mucosa was observed 2.5 hr following injection of dichlobenil (6, 12, 25 mg/kg). The synthetic GSH precursor N-acetyl-L-cysteine decreased both the dichlobenil-induced toxicity and the covalent binding, whereas N-acetyl-D-cysteine had no effect. No protective effects of the cyanide antidotes nitrite, thiosulfate, or superoxide dismutase on the dichlobenil-induced toxicity were observed. In mice given the GSH-depleting agent phorone and a subtoxic dose of dichlobenil (6 mg/kg), an extensive toxicity and an increased covalent binding in the olfactory mucosa were demonstrated. Autoradiography showed no change in the distribution of covalent [14C]dichlobenil binding to nontarget tissues of phorone-treated mice. In conclusion, the results demonstrate a relationship between the degrees of covalent binding, GSH depletion, and toxicity of dichlobenil in the olfactory mucosa. Hence, the level of GSH appears to be of importance for the dichlobenil-induced toxicity in the olfactory mucosa.     

Benzene Hemoglobin Adducts in Mice and Rats: Characterization of Formation and Physiological Modeling

Benzene Hemoglobin Adducts in Mice and Rats: Characterizationof Formation and Physiological Modeling. SUN, J. D., MEDINSKY,M. A., BIRNBAUM, L. S., LUCIER, G., AND HENDERSON, R. F. (1990).Fundam. Appl. Toxicol 15, 468–475. Benzene is a myelotoxinand a human leukemogen. Humans are exposed to this compound,both occupationally and environmentally. This study was conductedto determine whether formation of benzene-derived adducts withblood hemoglobin (Hb) can be used as a biomarker of exposureto benzene. B6C3F₁ mice and F344/N rats were given 0.1 to 10,000µmol [¹⁴C]benzene/kg body wt, orally. Twenty-four hourslater, animals were euthanized, and globin was isolated fromblood samples. The globin was analyzed by liquid scintillationspectrometry for the presence of [¹⁴C]benzene-de-rived adducts.Hb adduct formation was linear with respect to dose for amountsof up to 500 µmol [¹⁴C]benzene/kg body wt, for both rodentspecies. Within this linear dose-response range, mice formedadducts from [¹⁴C]benzene approximately 3.5 times less efficiently[0.022 ± 0.010 (pmol adducts/mg globin)/(µmol/kgbody wt dose)] than did rats [0.076 ± 0.014 (pmol adducts)/µmol/kg body wt dose)]. Benzene-derived Hb adducts alsoaccumulated linearly when mice and rats were given up to threedaily doses of 500 µmol [¹⁴C]benzene/kg body wt. Thesedata were used to develop a physiological model for benzene-derivedHb adduct formation. Both first-order and saturable pathwaysfor adduct formation were incorporated. The results showed thatthe model simulated the levels of Hb adducts in both mice andrats after oral exposures to benzene and predicted the levelsof Hb adducts present after inhalation exposure. These studiessuggest that Hb adducts might be useful biomarkers for humanexposures to benzene.     

In vivo studies on the target tissue metabolism,covalent binding,glutathione depletion,and toxicity of 4-ipomeanol in birds,species deficient in pulmonary enzymes for metabolic activation

Comparative studies on the target organ for covalent binding and toxicity by 4-ipomeanol, a furan derivative metabolically activated by and toxic to the nonciliated bronchiolar epithelial (Clara) cells in mammalian lung, were performed in birds. In birds, whose lungs lack the typical pulmonary ciliated and nonciliated bronchiolar cells and are deficient in enzymes necessary for the metabolic activation of 4-ipomeanol, the target organ for toxicity and covalent binding by 4-ipomeanol was the liver. Tissue damage was not observed in lungs or kidneys of either Japanese quail or roosters at any dose of 4-ipomeanol tested. Likewise, the levels of irreversibly bound 4-ipomeanol metabolites were highest in liver, with much lower levels in lungs, kidneys, and all other organs studied. In addition, toxic doses of 4-ipomeanol markedly depleted hepatic, but not pulmonary or renal glutathione in Japanese quail. The covalent binding of 4-ipomeanol metabolites and concomitant depletion of tissue glutathione in the liver were both time and dose dependent. In quail, over a dose range of 5 to 75 mg/kg, no dose threshold for covalent binding or hepatic damage which depended upon substantial glutathione depletion was observed. Pretreatment of quail with phenobarbital had no effect on liver cytochrome P-450 levels, nor did it have any effect on tissue covalent binding or toxicity by 4-ipomeanol. Although 3-methylcholanthrene pretreatment nearly tripled the microsomal cytochrome P-450 levels in quail liver, it had relatively little or no effect on the hepatic covalent binding or toxicity by 4-ipomeanol. Prior treatment with the drug metabolism inhibitor, piperonyl butoxide, markedly decreased the covalent binding, glutathione depletion, and the toxicity of 4-ipomeanol. Treatment of quail with diethylmaleate produced a dose-dependent depletion of hepatic, renal, and pulmonary glutathione, and it markedly increased hepatic covalent binding by 4-ipomeanol, but had no effect on renal or pulmonary covalent binding. Diethyl maleate markedly enhanced the hepatic necrosis by 4-ipomeanol. The results are consistent with the view that 4-ipomeanol is metabolized preferentially in quail liver to highly reactive metabolites which can be detoxified by glutathione. It appears unlikely however, that differences in detoxification via glutathione are the primary determinants of the remarkable tissue selectivity for covalent binding and toxicity of 4-ipomeanol in different animal species in vivo. The present studies support the view that the tissue selectivity of 4-ipomeanol resides primarily in the ability of the target tissue to rapidly metabolize 4-ipomeanol to its ultimate toxic metabolite(s). These investigations also suggest that the bird may be a potentially useful animal model in studies on the relationship of target organ toxicity to the formation of reactive metabolites by other chemicals requiring metabolic activation.     

Interactions between Bromobenzene Dose, Glutathione Concentrations, and Organ Toxicities in Single- and Multiple-Treatment Studies

Interactions between Bromobenzene Dose, Glutathione Concentrations,and Organ Toxicities in Single- and Multiple-Treatment Studies.KLUWE, W. M., MARONPOT, R. R., GREENWELL, A., AND HARRINGTON,F. (1984). Fundam. Appl. Toxicol. 4, 1019–1028. A singleoral dose of 4.0 mmol/kg bromobenzene transiently depleted hepaticand renal reduced nonprotein sulfhydryl group (NPS) concentrations,caused hepatocellular necrosis, and increased serum glutamic-pyruvictransaminase activity in male Fischer 344 rats. The depletionof NPS had partially reversed by 24 hr, and NPS concentrationswere approximately twice normal values by 48 hr post-treatmentWhen the effects of single and repeated (once dairy for 2, 4,or 10 days) treatments with 4.0 mmol/kg were compared, it wasapparent that the severity of hepatotoxicity lessened and thepercentage depletions of hepatic and renal NPS concentrationsdecreased with increasing length of bromobenzene treatment Therewere essentially no signs of toxicity following the tenth treatmentwith 4.0 mmol/kg. Single-treatment studies indicated the followingdose-response; 2.0 mmol/kg bromobenzene depleted liver NPS andwas hepatotoxic 0.5 mmol/ kg caused a lesser depletion of liverNPS and was not (overtly) hepatotoxic, and 0.0625 mmol/ kg wasthe maximum dose that did not deplete liver NPS. The responsesto single and multiple (ten) treatments with these representativedoses were compared. Liver injury was observed after a singlebut not after the tenth daily treatment with 2.0 mmol/kg. Boththe single and the tenth administrations of 2.0 mmol/kg depletedhepatic NPS, but the precentage of depletion was greater afterthe first than after the tenth dose. Liver injury was not detectedwith lower dose regimens. The patterns of NPS depletion in liverand kidney were similar after single or muliple (ten) treatments.The minimum NPS concentrations produced, however, were lowerafter single than after multiple treatments. The molar amountsof liver NPS depleted after the tenth treatment appeared tobe equivalent to or greater than those after the first but priorbromobenzene exposure resulted in a higher concentration oftissue NPS being present at the time of the final treatment.Thus, the minimum tissue concentrations of NPS were greaterafter multiple treatments than after single treatments, despitethe loss of equivalent amounts of NPS. It is concluded fromthese studies that repeated treatment produces resistance tobromobenzene hepatotoxicity. This protective adaptation maybe due to a chemically induced increase in liver glutathioneconcentration     

Species Differences in Kidney Toxicitity and Metabolic Activation of Tris(2,3-dibromopropyl)phosphate*

Species Differences in Kidney Toxicity and Metabolic Activationof Tris(2,3-dibromopropyl)phosphate. Sderlund, E.J., Nelson,S.D., von Bahr, C. and Dybing, E. Fundam. Appl. Toxicol. 2:187-194.The flame retardant tris(2,3-dibromopropyl)phosphate (Tris-BP)was studied for nephrotoxicity and covalent protein bindingin vivo to rat, mouse, hamster and guinea pig protein. In addition,Tris-BP mutagenicity to S. typhimurium TA 100 and covalent proteinbinding in vitro was determined with hepatic microsomes fromrats, mice, hamsters, guinea pigs and humans. Tris-BP causedacute tubular renal necrosis in rats at doses of 100 mg/kg i.p.and higher, whereas no clear evidence of renal damage was foundin mice, hamsters or guinea pigs at doses up to 500–1000mg/kg. After administration of radio-labelled Tris-BP to thevarious laboratory animals, all species showed similar levelsof covalent binding to proteins in liver and kidney, exceptfor the rat which had much higher amounts of radiolabel boundto kidney proteins, correlating with the observed species differencesin renal toxicity. Hepatic microsomes from all species, includingman, activated Tris-BP to mutagenic products, constitutive mutagenicrates in mice microsomes being highest. Microsomes from animalspretreated with phenobarbital were considerably more activethan control microsomes in mutagenic activation of Tris-BP.3-Methylcholanthrene-pretreatment only increased Tris-BP mutagenicitywith hamster and guinea pig liver microsomes. Clear interindividualdifferences in Tris-BP mutagenicity was noted with human hepaticmicrosomes. The rates of ³H-Tris-BP covalent protein bindingin vitro were of the same magnitude with hepatic microsomesfrom laboratory animals and humans. Phenobarbital-pretreatmentincreased binding rates 7-fold with rat and 2–3 fold withhamster and guinea pig microsomes, respectively, whereas noincrease was found with liver microsomes obtained from phenobarbital-pretreatedmice. 3-Methylchol-anthrene increased Tris-BP binding in vitroin hamsters and guinea pigs, but not in rats and mice. Covalentbinding of radiolabel from ³H-Tris-BP varied over a 3-fold rangeusing human hepatic microsomes.     

Covalent binding of acrylonitrile to specific rat liver glutathione S-transferases in vivo.

Acrylonitrile (AN) is an industrial vinyl monomer that is acutely toxic. When administered to rats, AN covalently binds to tissue proteins in a dose-dependent but nonlinear manner [Benz, F. W., Nerland, D. E., Li, J., and Corbett, D. (1997) Fundam. Appl. Toxicol. 36, 149-156]. The nonlinearity in covalent binding stems from the fact that AN rapidly depletes liver glutathione after which the covalent binding to tissue proteins increases disproportionately. The identity of the tissue proteins to which AN covalently binds is unknown. The experiments described here were conducted to begin to answer this question. Male Sprague-Dawley rats were injected subcutaneously with 115 mg/kg (2.2 mmol/kg) [2,3-(14)C]AN. Two hours later, the livers were removed, homogenized, and fractionated into subcellular components, and the radioactively labeled proteins were separated on SDS-PAGE. One set of labeled proteins was found to be glutathione S-transferase (GST). Specific labeling of the mu over the alpha class was observed. Separation of the GST subunits by HPLC followed by scintillation counting showed that AN was selective for subunit rGSTM1. Mass spectral analysis of tryptic digests of the GST subunits indicated that the site of labeling was cysteine 86. The reason for the high reactivity of cysteine 86 in rGSTM1 was hypothesized to be due to its potential interaction with histidine 84, which is unique in this subunit.     

Effects of the herbicide chlorthiamid on the olfactory mucosa

Chlorthiamid (2,6-dichlorothiobenzamide) and its major metabolite 2,6-dichlorobenzonitrile are olfactory toxicants with a high in vivo covalent binding in the olfactory mucosa of mice. This study showed that the cytochrome P450 (P450) inhibitors, metyrapone and sodium-diethyldithiocarbamate, abolished the chlorthiamid-induced toxicity (12 mg/kg; 0.06 mmol/kg) in C57Bl/6 mice suggesting a P450-dependent toxicity. Incubation of [¹⁴C]-labelled chlorthiamid with rat olfactory microsomes showed a low NADPH-dependent oxidative covalent binding which was only 3-fold higher than that in liver microsomes. Thus the results do not support a major in situ metabolic activation of chlorthiamid and it is suggested that metabolic activation of the major chlorthiamid metabolite (2,6-dichlorobenzonitrile) is responsible for most of the covalent binding and toxicity of chlorthiamid at this site in vivo. Thiobenzamide (16 mg/kg; 0.12 mmol/kg), a dechlorinated chlorthiamid-analog, induced no marked morphological changes in the olfactory mucosa demonstrating that chlorines in the 2,6-position are important for the chlorthiamid-induced toxicity at this site.     

Selective covalent binding of acrylonitrile to Cys 186 in rat liver carbonic anhydrase III in vivo

Covalent binding of reactive chemical species to tissue proteins is a common, but poorly understood, mechanism of toxicity. Identification of the proteins and the specific amino acid residues within the proteins that are chemically modified will aid our understanding of the toxification/detoxification mechanisms involved in covalent binding. Acrylonitrile (AN) is a commercial vinyl monomer that is acutely toxic and readily binds to tissue proteins. Total covalent binding of AN to tissue proteins is highly correlated with acute toxicity. Two-dimensional PAGE and autoradiography were used to locate proteins in male rat liver cytosol that are radiolabeled following administration of [2,3-(14)C]AN in vivo. Four intensely labeled spots were prominent in the autoradiogram and formed an apparent "charge-train" at approximately 30 kDa. Tryptic peptide mapping by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS was used to identify all of the spots as carbonic anhydrase III (CAIII). HPLC of the tryptic digests combined with MALDI-TOF MS was used to localize the radiolabel to tryptic fragment T22 containing amino acids 171-187. This tryptic fragment contains two Cys residues (Cys181 and Cys186) in the rat CAIII sequence. Electrospray ionization ion-trap MS was used to sequence the peptide and establish that only Cys186 was labeled. Thus, although AN is considered to be highly reactive, our data indicate that it does not react indiscriminately with rat CAIII but rather is selective for one out of five Cys residues. Rat liver CAIII has previously been shown to protect cells against oxidative stress. Our data suggest that CAIII is also capable of scavenging reactive xenobiotics and may help prevent covalent binding to more critical macromolecules.     

Renal damage, metabolism and covalent binding following administration of the nephrotoxicant N-(3,5-dichlorophenyl)succinimide (NDPS) to male Fischer 344 rats.

In vivo metabolism, nephrotoxicity and covalent binding to proteins were evaluated in male Fischer 344 rats that received [2,3-¹⁴C]-N-(3,5-dichlorophenyl)succinimide (¹⁴C-NDPS). Some animals were pretreated with the enzyme inducer phenobarbital (PB, 80 mg/kg per day, for 3 days, i.p. in saline) prior to receiving a non-nephrotoxic dose of ¹⁴C-NDPS (0.2 mmol/kg, i.p. in corn oil). Other rats were pretreated with the cytochrome P450 inhibitor 1-aminobenzotriazole (ABT, 100 mg/kg, 1 h prior to NDPS, i.p. in saline) before administration of a non-toxic or a toxic dose (0.2 or 0.6 mmol/kg, respectively, i.p. in corn oil) of ¹⁴C-NDPS. Non-pretreated animals received either dose of ¹⁴C-NDPS, but did not receive PB or ABT. All rats were sacrificed 6 h after administration of ¹⁴C-NDPS. Nephrotoxicity was monitored by measuring urine volume, urine protein concentrations, blood urea nitrogen levels, and kidney weights. The NDPS metabolic profile in tissue, blood, and urine was analyzed by HPLC. Covalent binding of ¹⁴C-NDPS-derived radioactivity to tissue proteins was also measured. Compared with non-pretreated rats, PB-pretreatment potentiated the toxicity of the non-toxic dose of ¹⁴C-NDPS. In contrast, ABT-pretreatment protected the rats against NDPS nephrotoxicity. The amount of N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA), an oxidative, nephrotoxic metabolite of NDPS, was elevated in kidney homogenates and urine by PB-pretreatment (0.2 mmol/mg NDPS). ABT pretreatment inhibited NDPS metabolism at both doses. Covalent binding of ¹⁴C-NDPS (0.2 mmol/kg)-derived radioactivity to renal and plasma proteins was higher in the PB-pretreated rats than in the non-pretreated animals. In contrast, ABT-pretreatment partially inhibited covalent binding at both doses of ¹⁴C-NDPS. Our results suggest that there is a relationship between oxidative metabolism of NDPS, covalent binding of an NDPS metabolite to renal proteins, and NDPS-induced nephrotoxicity in rats.     

Renal damage, metabolism and covalent binding following administration of the nephrotoxicant N-(3,5-dichlorophenyl)succinimide (NDPS) to male Fischer 344 rats

In vivo metabolism, nephrotoxicity and covalent binding to proteins were evaluated in male Fischer 344 rats that received [2,3-¹⁴C]-N-(3,5-dichlorophenyl)succinimide (¹⁴C-NDPS). Some animals were pretreated with the enzyme inducer phenobarbital (PB, 80 mg/kg per day, for 3 days, i.p. in saline) prior to receiving a non-nephrotoxic dose of ¹⁴C-NDPS (0.2 mmol/kg, i.p. in corn oil). Other rats were pretreated with the cytochrome P450 inhibitor 1-aminobenzotriazole (ABT, 100 mg/kg, 1 h prior to NDPS, i.p. in saline) before administration of a non-toxic or a toxic dose (0.2 or 0.6 mmol/kg, respectively, i.p. in corn oil) of ¹⁴C-NDPS. Non-pretreated animals received either dose of ¹⁴C-NDPS, but did not receive PB or ABT. All rats were sacrificed 6 h after administration of ¹⁴C-NDPS. Nephrotoxicity was monitored by measuring urine volume, urine protein concentrations, blood urea nitrogen levels, and kidney weights. The NDPS metabolic profile in tissue, blood, and urine was analyzed by HPLC. Covalent binding of ¹⁴C-NDPS-derived radioactivity to tissue proteins was also measured. Compared with non-pretreated rats, PB-pretreatment potentiated the toxicity of the non-toxic dose of ¹⁴C-NDPS. In contrast, ABT-pretreatment protected the rats against NDPS nephrotoxicity. The amount of N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA), an oxidative, nephrotoxic metabolite of NDPS, was elevated in kidney homogenates and urine by PB-pretreatment (0.2 mmol/mg NDPS). ABT pretreatment inhibited NDPS metabolism at both doses. Covalent binding of ¹⁴C-NDPS (0.2 mmol/kg)-derived radioactivity to renal and plasma proteins was higher in the PB-pretreated rats than in the non-pretreated animals. In contrast, ABT-pretreatment partially inhibited covalent binding at both doses of ¹⁴C-NDPS. Our results suggest that there is a relationship between oxidative metabolism of NDPS, covalent binding of an NDPS metabolite to renal proteins, and NDPS-induced nephrotoxicity in rats.     

The Effect of 2-(o-Cresyl)-4H-1:3:2-benzodioxaphosphorin-2-oxide on Tissue Cholinesterase and Carboxylesterase Activities of the Rat

The dose-response (0.1 to 1000 mg/kg sc) effects of 2-(o-cresyl)-4H-l:3:2-benzodioxaphosphorin-2-oxide(CBDP; a melabolite of the organophosphorus compound tri-o-cresylphosphareon total cholinesterase (ChE) and carboxylesterase (CaE) activitiesin tissues from the rat were examined. Doses of CBDP greaterthan 1.0 mg/kg inhibited CaE activity maximally (>99%) inplasma and lung, two important sites for detoxification of organophosphorustoxicants. A biphasic dose-dependent inhibition of ChE activitywas seen in all tissues; the ED5O values showed a differenceof two orders of magnitude between the first and the secondphases of the dose-response curves. CBDP inhibited the bloodesterases in the order plasma CaE plasma ChE red blood cell(RBC) ChE. The biphasic dose-response curve and preferentialinhibition of the blood esterases may reflect the inhibitionof butyryicholinesterase in preference to acetylcholinesterasein these tissues. At doses of CBDP below 1.0 mg/kg, plasma,RBC, and brain regional ChE activities were inhibited by lessthan 10%, whereas at doses above 2.0 mg/kg, ChE activities wereinhibited substantially (up to 80% in plasma, up to 60% in RBC,and greater than 90% in brain regions). On the basis of theseresults, a dose of CBDP between 1.0 and 2.0 mg/kg should proveuseful as a pretreatment for studies of OP toxicity in the rat.     

Potential Application of Benzo(a)Pyrene-Associated Adducts (Globin or Lipid) as Blood Biomarkers for Target Organ Exposure and Human Risk Assessment

In order to investigate the potential application of blood biomarkers as surrogate indicators of carcinogen–adduct formation in target-specific tissues, temporal formation of benzo[a]pyrene (BaP)-associated DNA adducts, protein adducts, or lipid damage in target tissues such as lung, liver, and kidney was compared with globin adduct formation or plasma lipid damage in blood after continuous intraperitoneal (ip) injection of [³H]BaP into female ICR mice for 7 d. Following treatment with [³H]BaP, formation of [³H]BaP–DNA or –protein adducts in lung, liver, and kidney increased linearly, and persisted thereafter. This finding was similar to the observed effects on globin adduct formation and plasma lipid damage in blood. The lungs contained a higher level of DNA adducts than liver or kidneys during the treatment period. Further, the rate of cumulative adduct formation in lung was markedly greater than that in liver. Treatment with a single dose of [³H]BaP indicated that BaP–globin adduct formation and BaP–lipid damage in blood reached a peak 48 h after treatment. Overall, globin adduct formation and lipid damage in blood were significantly correlated with DNA adduct formation in the target tissues. These data suggest that peripheral blood biomarkers, such as BaP–globin adduct formation or BaP–lipid damage, may be useful for prediction of target tissue-specific DNA adduct formation, and for risk assessment after exposure.     

Correlation of a specific mitochondrial phospholipid-phosgene adduct with chloroform acute toxicity

The dose and time dependence of formation of a specific adduct between mitochondrial phospholipid and phosgene have been determined in the liver of Sprague-Dawley (SD) rats as well as in the liver and kidney of B6C3F1 mice after dosing with chloroform. Rats were induced with phenobarbital or non-induced. Determination of tissue glutathione (GSH) and of serum markers of hepatotoxicity and nephrotoxicity was also carried out. With dose-dependence experiments, a strong correlation between the formation of the specific phospholipid adduct, GSH depletion and organ toxicity could be evidenced in all the organs studied. With non-induced SD rats, no such effects could be induced up to a dose of 740 mg/kg. Time-course studies with B6C3F1 mice indicated that the specific adduct formation took place at very early times after chloroform dosing and was concurrent with GSH depletion. The adduct formed during even transient GSH depletion (residual level: 30% of control) and persisted after restoration of GSH levels. Following a chloroform dose at the hepatotoxicity threshold (150 mg/kg), the elimination of the adduct in the liver occurred within 24 h and correlated with the recovery of ALT, which was slightly increased (12 times) after treatment. Following a moderately nephrotoxic dose (60 mg/kg), the renal adduct persisted longer than 48 h, when a 100% increase in blood urea nitrogen and a 40% increase in serum creatinine indicated the onset of organ damage. The formation of the adduct in the liver mitochondria of B6C3F1 mice was associated with the decrease of phosphatidyl-ethanolamine (PE), in line with previous results in rat liver indicating that the adduct results from the reaction of phosgene with PE. The adduct levels implicated the reaction of phosgene with about 50% PE molecules in the liver mitochondrial membrane of phenobarbital-induced SD rats and of about 10% PE molecules of the inner mitochondrial membrane of the liver of B6C3F1 mice. The association of this adduct with the toxic effects of chloroform makes it a very good candidate as the primary critical alteration in the sequence of events leading to cell death caused by chloroform.     

Comparison of the distribution of three bisphosphonates in mice

The distribution of approximately equipotent doses of 14C-labelled clodronate (0.1 mmol/kg), etidronate (0.1 mmol/kg), and amidronate (0.015 mmol/kg), and also an equimolar dose of amidronate (0.1 mmol/kg), was studied in mice by measuring the 14C-activities in various tissues up to 360 days after a single intravenous injection. With the higher dose of amidronate the distribution could be, however, monitored only for 7 days because of the toxicity of the drug. The results indicate that there are major differences in the deposition of the bisphosphonates into soft tissues, while the disappearance from plasma and incorporation into bone are quite similar in terms of percentage of dose per g of tissue. However, the binding capacity of bone for clodronate and etidronate is many times greater than that for amidronate expressed as nmol of the drug per g of tissue. Amidronate deposits in the spleen and liver of mice, when injected in saline, whereas clodronate and etidronate do not. This agrees with the suggestion that amidronate, but not the other two, can interfere with the mononuclear phagocyte system of spleen and liver.