Acetaldehyde and retinaldehyde-metabolizing enzymes in colon and pancreatic cancers.

Colorectal cancer (CRC) and pancreatic cancer are two very significant contributors to cancer-related deaths. Chronic alcohol consumption is an important risk factor for these cancers. Ethanol is oxidized primarily by alcohol dehydrogenases to acetaldehyde, an agent capable of initiating tumors by forming adducts with proteins and DNA. Acetaldehyde is metabolized by ALDH2, ALDH1B1, and ALDH1A1 to acetate. Retinoic acid (RA) is required for cellular differentiation and is known to arrest tumor development. RA is synthesized from retinaldehyde by the retinaldehyde dehydrogenases, specifically ALDH1A1, ALDH1A2, ALDH1A3, and ALDH8A1. By eliminating acetaldehyde and generating RA, ALDHs can play a crucial regulatory role in the initiation and progression of cancers. ALDH1 catalytic activity has been used as a biomarker to identify and isolate normal and cancer stem cells; its presence in a tumor is associated with poor prognosis in colon and pancreatic cancer. In summary, these ALDHs are not only biomarkers for CRC and pancreatic cancer but also play important mechanistic role in cancer initiation, progression, and eventual prognosis.

Glutathione defense mechanism in liver injury: insights from animal models.

Glutathione (GSH) is the most abundant cellular thiol antioxidant and it exhibits numerous and versatile functions. Disturbances in GSH homeostasis have been associated with liver diseases induced by drugs, alcohol, diet and environmental pollutants. Until recently, our laboratories and others have developed mouse models with genetic deficiencies in glutamate-cysteine ligase (GCL), the rate-limiting enzyme in the GSH biosynthetic pathway. This review focuses on regulation of GSH homeostasis and, specifically, recent studies that have utilized such GSH-deficient mouse models to investigate the role of GSH in liver disease processes. These studies have revealed a differential hepatic response to distinct profiles of hepatic cellular GSH concentration. In particular, mice engineered to not express the catalytic subunit of GCL in hepatocytes [Gclc(h/h) mice] experience almostcomplete loss of hepatic GSH (to 5% of normal) and develop spontaneous liver pathologies characteristic of various clinical stages of liver injury. In contrast, mice globally engineered to not express the modifier subunit of GCL [Gclm⁻/⁻ mice] show a less severe hepatic GSH deficit (to ≈15% of normal) and exhibit overall protection against liver injuries induced by a variety of hepatic insults. Collectively, these transgenic mouse models provide interesting new insights regarding pathophysiological functions of GSH in the liver.

Comparative metabolism, covalent binding and toxicity of BHT congeners in rat liver slices.

The metabolism, covalent binding and hepatotoxicity of butylated hydroxytoluene (BHT, 4-methyl-2,6-di-t-butylphenol) and two congeners (E-BHT, 4-ethyl-2,6-di-t-butylphenol; I-BHT, 4-isopropyl-2,6-di-t-butylphenol) were compared using precision-cut liver slices prepared from phenobarbital (PB)-treated male Sprague-Dawley rats. At equimolar concentrations (1 mM) BHT was the most toxic of the three compounds, causing an 80% decrease in cell viability over a 6 h incubation period. E-BHT was intermediate in toxicity while the isopropyl derivative was relatively nontoxic. Intracellular glutathione levels decreased prior to the onset of cytotoxicity. The cytochrome P450 inhibitor metyrapone completely inhibited the toxicity of all three compounds. The rates of metabolism of the three compounds to glutathione conjugates were compared in both PB-treated microsomes and PB-induced liver slices. In both models, the rate of formation was greatest for BHT, followed by E-BHT and I-BHT. Synthetic quinone methides (QMs) were prepared from each parent phenol and the rates of reactivity with three nucleophiles (water, methanol and glutathione) were compared. With each nucleophile, BHTQM was the most reactive, while I-BHTQM was the least reactive. Finally, covalent binding to protein was assessed in two ways. First, alkylation of an isolated model protein (bovine insulin) was measured in a microsomal enzyme activation system by mass spectrometry. Incubations with BHT produced the greatest extent of protein alkylation, followed by E-BHT, while no alkylation was observed with I-BHT. In the second system, covalent binding to cellular protein was assessed in rat liver PB microsomes and tissue slices by Western blotting using an antibody specific for the tert-butylphenol portion of the compounds. Binding was greatest for BHT, intermediate for E-BHT and could not be detected for I-BHT. The alkylation pattern for E-BHT was strikingly similar to that of BHT, suggesting that both compounds bound similar proteins. In summary, our results suggest that for hindered phenols such as BHT, increasing the length of the 4-alkyl substituent retards the rate of formation of reactive intermediates, significantly reduces the electrophilicity of the reactive intermediate, and greatly reduces the amount but not the selectivity of covalent binding to cellular protein, thereby reducing the toxicity of the parent compound.

Growth inhibition in G(1) and altered expression of cyclin D1 and p27(kip-1 )after forced connexin expression in lung and liver carcinoma cells.

Gap junctional intercellular communication (GJIC) and connexin expression are frequently decreased in neoplasia and may contribute to defective growth control and loss of differentiated functions. GJIC, in E9 mouse lung carcinoma cells and WB-aB1 neoplastic rat liver epithelial cells, was elevated by forced expression of the gap junction proteins, connexin43 (Cx43) and connexin32 (Cx32), respectively. Transfection of Cx43 into E9 cells increased fluorescent dye-coupling in the transfected clones, E9-2 and E9-3, to levels comparable to the nontransformed sibling cell line, E10, from which E9 cells originated. Transduction of Cx32 into WB-aB1 cells also increased dye-coupling in the clone, WB-a/32-10, to a level that was comparable to the nontransformed sibling cell line, WB-F344. The cell cycle distribution was also affected as a result of forced connexin expression. The percentage of cells in G(1)-phase increased and the percentage in S-phase decreased in E9-2 and WB-a/32-10 cells as compared to E9 and WB-aB1 cells. Concomitantly, these cells exhibited changes in G(1)-phase cell cycle regulators. E9-2 and WB-a/32-10 cells expressed significantly less cyclin D1 and more p27(kip-1) protein than E9 and WB-aB1 cells. Other growth-related properties (expression of platelet-derived growth factor receptor-beta, epidermal growth factor receptor, protein kinase C-alpha, protein kinase A regulatory subunit-Ialpha, and production of nitric oxide in response to a cocktail of pro-inflammatory cytokines) were minimally altered or unaffected. Thus, enhancement of connexin expression and GJIC in neoplastic mouse lung and rat liver epithelial cells restored G(1) growth control. This was associated with decreased expression of cyclin D1 and increased expression of p27(kip-1), but not with changes in other growth-related functions.

Spontaneous hydrolysis of 4-trifluoromethylphenol to a quinone methide and subsequent protein alkylation.

4-Trifluoromethylphenol (4-TFMP) was cytotoxic to precision-cut rat liver slices as indicated by loss of intracellular potassium. Intracellular glutathione levels decreased and fluoride ion levels increased in a time and concentration-dependent manner. The cytotoxicity of 4-TFMP did not appear to be due to the release of fluoride, however, since equimolar concentrations of sodium fluoride or potassium fluoride were not toxic. The ortho isomer (2-TFMP), which had a threefold slower rate of fluoride release, was much less toxic to liver slices. In incubations without slices, 4-TFMP spontaneously hydrolyzed in aqueous buffer at physiological pH to form 4-hydroxybenzoic acid via a quinone methide intermediate. The quinone methide was trapped by the addition of glutathione. Analysis of the glutathione adduct indicated that all of the fluorine atoms were lost during the hydrolysis, yielding a cresol derivative with the glutathione moiety attached to a benzylic carbonyl group. The glutathione conjugate was the primary product formed at low alkylphenol/glutathione ratios; however, at higher 4-TFMP concentrations additional unidentified products were observed. 4-TFMP also inhibited the in vitro enzyme activity of purlfied glyceraldehyde-3-phosphate dehydrogenase, a sulfhydryl-dependent enzyme, in a time and concentration-dependentmanner. Loss of thiol residues closely paralleled the loss in enzyme activity. The coaddition of glutathione prevented 4-TFMP-induced loss of enzyme activity. The cytotoxicity of 4-TFMP therefore appears to be due to spontaneous quinone methide formation and subsequent alkylation of cellular macromolecules.

Domestic violence and primary care. Attitudes, practices, and beliefs.

OBJECTIVE: To assess the attitudes and beliefs of the primary care provider team (physicians, physician assistants, nurses, and medical assistants) toward the identification and management of abused patients and perpetrators of domestic violence (DV). DESIGN: Survey of the health care team using a confidential questionnaire. SETTING AND SUBJECTS: Five primary care clinics with 240 providers at a large urban health maintenance organization. RESULTS: The response rate was 86% (206 respondents). Fifty percent of clinicians and 70% of nurses/assistants believed that the prevalence of DV in their practice was 1% or loss; 1 in 10 clinicians and nearly half of nurses/assistants had never identified an abused person; 45% of clinicians never or seldom asked about DV when examining injured patients; and all participants were much less confident in asking about DV than about smoking or consuming alcohol. Twenty-five percent believed the abused person's personality led to the violence; 28% believed they did not have strategies to help abused persons; and 20% were concerned for their personal safety in discussing DV. Only 10% believed they had management information, but 77% had not attended any educational programs on DV in the past year. CONCLUSIONS: This study provides important information about current knowledge, attitudes, and beliefs of health care providers toward the diagnosis and management of DV. This information should prove useful to all who attempt to design clinical strategies and educational programs to address this issue.

Does the face protect the brain? A case-control study of traumatic brain injury and facial fractures.

BACKGROUND: The relationship between facial fractures and traumatic brain injury is controversial. Some studies show an increased risk of brain injury with the presence of facial fractures while others claim that facial fractures protect against brain injury. OBJECTIVE: To examine the association between facial fractures and traumatic brain injuries. DESIGN: Case-control study. SETTING: Subjects were recruited from the emergency departments of 7 hospitals in the Seattle, Wash, area. PATIENTS: Three thousand eight hundred forty-nine injured bicyclists and 5 scene deaths were identified from March 1, 1992, to August 31, 1994, with complete data available on 3388 bicyclists. INTERVENTIONS: None. RESULTS: The study group was composed of 1602 cases with injuries to the head, face, or brain and 1540 control subjects. There were 203 bicyclists with traumatic brain injuries, of whom 62 had an identifiable intracranial injury and 141 suffered a concussion. A total of 81 patients sustained facial fractures. The odds ratio for the risk of intracranial injury associated with facial fractures after adjustment for significant confounders was 9.9 (95% confidence interval, 5.1-19.3). The effect was less strong but still present when all traumatic brain injuries including concussions were considered (odds ratio, 2; 95% confidence interval, 1.1-3.7). No association was found for concussion only. CONCLUSIONS: This study demonstrates no evidence that facial fractures help prevent traumatic brain injury. Data indicate that facial fractures are markers for increased risk of brain injury.

Cytokine-induced nitric oxide formation in normal but not in neoplastic murine lung epithelial cell lines.

Cytomix, a mixture of interferon-gamma, tumor necrosis factor-alpha, and interleukin-1beta, induces nitric oxide (NO) production in lung epithelial cell lines. It is not known whether neoplastic transformation alters a cell's ability to form NO in response to cytokines. The present study investigated NO formation in two murine lines of immortalized "normal" (nontumorigenic) lung epithelial cells of alveolar type II origin, E10 and C10, and their sibling spontaneous transformants, E9 and A5. Nontumorigenic cells elaborated much more NO after cytomix exposure than did their tumorigenic counterparts. NO production was prevented by inhibiting protein synthesis and NO synthase and attenuated by dexamethasone. Northern and Western blot analyses of inducible NO synthase (iNOS) demonstrated cytomix-induced induction of iNOS only in nontumorigenic cells. The deficiency in NO production in tumorigenic cells was not associated with reduced iNOS mRNA stability or with differences in cytomix-induced nuclear factor-kappaB activation. Although cytomix caused a greater production of NO in E10 cells than in E9 cells, the same treatment induced equivalent proliferation in both cell lines. These results indicate a specific deficiency in cytokine-induced NO synthesis in transformed murine lung epithelial cells relative to their normal progenitor cells and provide a model for investigating iNOS regulation.

Comparative toxicity of eugenol and its quinone methide metabolite in cultured liver cells using kinetic fluorescence bioassays.

Comparative kinetic analyses of the mechanisms of toxicity of the alkylphenol eugenol and its putative toxic metabolite (quinone methide, EQM) were carried out in cultured rat liver cells (Clone 9, ATCC) using a variety of vital fluorescence bioassays with a Meridian Ultima laser cytometer. Parameters monitored included intracellular GSH and calcium levels ([Ca2+]i), mitochondrial and plasma membrane potentials (MMP and PMP), intracellular pH, reactive oxygen species (ROS) generation, and gap junction-mediated intercellular communication (GJIC). Cells were exposed to various concentrations of test compounds (1 to 1000 microM) and all parameters monitored directly after addition at 15 s intervals for at least 10 min. Eugenol depleted intracellular GSH, inhibited GJIC and generation of ROS, and had a modest effect on MMP at concentrations of 10 to 100 microM. At high concentrations (1000 microM), eugenol also affected [Ca2+]i, PMP, and pH. Effects of EQM were seen at lower concentrations (1 to 10 microM). The earliest and most potent effects of either eugenol or EQM were seen on GSH levels and GJIC. Coadministration of glutathione ethyl ester enhanced intracellular GSH levels by almost 100% and completely protected cells from cell death caused by eugenol and EQM. These results suggest that eugenol mediates its hepatotoxic effects primarily through depletion of cytoprotective thiols and interference in thiol-dependent processes such as GJIC. Furthermore, our results support the hypothesis that the toxic effects of eugenol are mediated through its quinone methide metabolite.

Differential susceptibility of tracheal contraction to nonadrenergic noncholinergic relaxation.

Studies of nonadrenergic noncholinergic inhibitory (NANCi) nerve-induced relaxations routinely examine relaxations in airway tissue in which tone has been established. Little is known about the ability of NANCi nerve stimulation to prevent airway smooth muscle contraction. The present study compares the capacity of NANCi nerve stimulation to prevent or reverse airway smooth muscle contraction. NANCi nerves in the trachea from ovalbumin-sensitized guinea pigs were subjected to electrical field stimulation (EFS, 10 Hz, 0.3 ms, 10 V, 35 min) initiated before or after induction of tone with antigen or histamine. In tissues precontracted with histamine or antigen, EFS elicited a rapid relaxation which peaked within the first 5 min and stabilized by 20 to 35 min. The peak relaxation was smaller in tissues precontracted with antigen, an effect that was not prevented by tissue treatment with a nitric oxide synthase inhibitor. In contrast, the stabilized level of NANCi relaxation did not differ between histamine- or antigen-contracted tissues. Activation of NANCi nerves prior to induction of tone also resulted in inhibition of the contractile actions of histamine and antigen. However, the stabilized level of tone induced by a contractile agonist added after initiation of EFS was greater than the stabilized tone caused by EFS in tissues already contracted with the same agonist. Relaxations elicited by S-nitrosoglutathione were reduced in antigen-precontracted tissues whereas vasoactive intestinal peptide-induced relaxant responses were similar in antigen- and histamine-precontracted tissues. Results of this study suggest that NANCi nerve activation is more effective at relaxing established airway smooth muscle tone than at preventing airway smooth muscle contraction. Further, the results suggest that the difference in NANCi activity in antigen-precontracted tissues cannot be ascribed solely to reductions in the nitric oxide-dependent component of the response.

Absence of tachykinin involvement in leukotriene D4 and antigen-induced contraction of guinea pig isolated bronchus.

In guinea pig airways, contractions induced by leukotriene D4 or antigen are thought to be mediated primarily by an action of the agonist or of released mast cell-derived mediators directly on the airway smooth muscle cell. An indirect contractile action mediated by endogenous tachykinins also has been described for both of these stimuli. The present study evaluated the contribution of endogenous tachykinins to ovalbumin- and leukotriene D4-induced contractions in the guinea pig bronchus by modulating the concentrations of tachykinins within the tissues and by using neurokinin receptor antagonists. Acute depletion of tachykinins with capsaicin had no effect on responses elicited by either stimulus. Similarly, tetrodotoxin treatment failed to influence leukotriene D4-induced contractions. Inhibitors of neutral endopeptidase (thiorphan) and angiotensin-converting enzyme (lisinopril) enhanced neurally mediated tachykininergic responses and potentiated leukotriene D4. The latter effect persisted in the presence of tetrodotoxin or the neurokinin antagonists CP99994 and SR48968 and in tissues treated acutely with capsaicin. The potentiation was absent, however, from bronchi incubated with L-cysteine. Ovalbumin-induced contractions were unaltered by inhibition of neutral endopeptidase and angiotensin-converting enzyme. These observations suggest that tachykinins are not involved in mediation of leukotriene D4- or antigen-induced contractions of the guinea pig bronchus. The ability of protease inhibitors to potentiate leukotriene D4 but not antigen-induced responses is therefore ascribed to inhibition of bioinactivation of leukotriene D4 to leukotriene E4.

Metabolism and toxicity of 4-hydroxyphenylacetone in rat liver slices: comparison with acetaminophen.

Acetaminophen is oxidized by cytochrome P450 to a reactive quinone imine, N-acetyl-p-benzoquinone imine, which is thought to be responsible for its hepatotoxic effects. 4-Hydroxyphenylacetone (4-HPA) is a structural analog of acetaminophen in which the amine group is replaced by a methylene group. Following a similar metabolic pathway, 4-HPA would be oxidized to form a reactive quinone methide intermediate. We compared the metabolism and toxicity of 4-HPA and acetaminophen in liver microsomes and precision-cut liver slices from male Sprague-Dawley rats. Both 4-HPA and acetaminophen formed glutathione conjugates in microsomal incubations. 4-HPA formed diastereomeric glutathione conjugates, which is consistent with the formation of an intermediate quinone methide. The rate of conjugate formation with 4-HPA was 8.5-fold greater than that with acetaminophen. In rat liver slices a concentration of 5 mM 4-HPA killed approximately 50% of hepatocytes after 6 hr of incubation, whereas acetaminophen was not toxic at concentrations up to 50 mM. N-Acetylcysteine protected slices from 4-HPA-induced toxicity, whereas phenobarbital enhanced metabolism and toxicity. In summary, 4-HPA is more hepatotoxic than acetaminophen, and this may be the result of differences in the metabolic rate and/or the type of reactive intermediate formed.

Studies on the mechanism of hepatotoxicity of 4-methylphenol (p-cresol): effects of deuterium labeling and ring substitution.

We recently observed that 4-methylphenol (p-cresol) is toxic to rat liver tissue slices. A possible mechanism involves biotransformation of 4-methylphenol to a reactive quinone methide intermediate which covalently binds to cellular macromolecules and elicits cytotoxicity. In order to obtain further evidence for this proposed mechanism, we studied the effects of deuterium-labeled 4-methylphenol (4-[alpha, alpha, alpha-d3]-methylphenol), and the presence of various ring substituents, on the metabolism and toxicity of 4-methylphenol in precision cut liver slices prepared from male Sprague-Dawley rats. Deuterium-labeled 4-methylphenol was significantly less toxic than the parent compound in rat liver slices (LC50 = 3.36 vs. 1.31 mM, respectively). In addition, the deuterium-labeled compound was metabolized to a reactive intermediate (measured as glutathione conjugate formation) at a slower rate than that of 4-methylphenol in both liver slices and liver microsomal incubations. The presence of electron withdrawing substituents (2-chloro or 2-bromo) markedly enhanced both metabolism and toxicity, with the exception of 2,6-dibromocresol, which was similar to cresol in terms of rate of metabolism and toxicity. Conversely, the presence of electron donating substituents (2-methoxy, 2-methyl or 2,6-dimethyl) diminished metabolism and toxicity. In addition, methylation of the hydroxyl group to form 4-methylanisole, greatly reduced toxicity. These results support the hypothesis that the toxicity of 4-methylphenol is dependent on the formation of a reactive quinone methide intermediate.

HPLC and 31P NMR characterization of the reaction between antitumor platinum agents and the phosphorothioate chemoprotective agent S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721).

In prior studies, we examined the effects of the radioprotective and chemoprotective agent WR-2721 [S-2-(3-aminopropylamino)ethylphosphorothioic acid] on the in vivo biotransformation of the cisplatin [cis-diamminedichloroplatinum(II)] analog ormaplatin [(d,I)trans-1,2-diaminocyclohexanetetrachloroplatinum(IV), Pt(dach)Cl4, (formerly called tetraplatin)]. Those data suggested that a direct interaction between WR-2721 and ormaplatin and/or the corresponding Pt(II) drug, Pt(dach)Cl2, may be occurring in vivo. This would be in contrast to the generally accepted hypothesis that WR-2721 is a prodrug that must first be converted by alkaline phosphatase to a free thiol compound, WR-1065, before any appreciable reactivity would be evident. However, the major biotransformation product observed in the peritoneal fluid, plasma, and all tissues was Pt(dach)(WR-1065). We report here on further investigations into the in vitro reactivity of Pt(dach) compounds with WR-2721 and WR-1065. Separation of reaction products resulting from incubation of Pt(dach)(malonato) with either WR-2721 or WR-1065 under physiological conditions gave profiles that were indistinguishable by reverse phase HPLC and cation exchange HPLC at two different pHs. 31P NMR characterization of the dephosphorylation of WR-2721 revealed essentially no loss of inorganic phosphate for up to 24 hr when incubated in unbuffered water at 30 degrees. In contrast, when incubated with a 1:1 molar ratio of cisplatin under the same conditions, the WR-2721 signal was decreased markedly in the first 5 min, and had disappeared almost completely by 1 hr. The signal corresponding to inorganic phosphate increased in parallel to the decrease in the WR-2721 signal. No intermediate formation of a complex containing both platinum and phosphate could be detected at any time. These data suggest that the reaction between WR-2721 and platinum complexes results in rapid dephosphorylation of WR-2721, and, consequently, that the reaction products formed with either WR-2721 or WR-1065 and Pt(II) complexes are identical.

Effect of the chemoprotective agent WR-2721 on disposition and biotransformations of ormaplatin in the Fischer 344 rat bearing a fibrosarcoma.

The effects of the phosphorothioate agent, WR-2721, have been investigated with respect to the biotransformations of ormaplatin in the Fischer 344 rat bearing a transplanted fibrosarcoma. A number of different paradigms of dosing route and schedule for the administration of the two agents have been investigated. In the first group of experiments, WR-2721 (200 mg/kg, i.p.) was administered 30 min before ormaplatin (12.5 mg/kg, i.p.), and then peritoneal fluid, plasma, and tissues were harvested at 30 min after the ormaplatin administration. Our results suggest that a significant interaction between WR-2721 and ormaplatin is occurring in the peritoneal cavity. The interaction was evident in terms of both effects on distribution and disposition of total platinum and in alterations of the profiles of biotransformation products formed in the various tissues and fluids. Plasma protein binding of ormaplatin was decreased by 50% in the presence of WR-2721. Total platinum in the spleen was decreased by 66% and in the liver by 50%. There were no trends among the findings that would indicate any selectivity between tumor and nontumor tissue with respect to the effects of WR-2721 on the parameters measured. Subsequent investigations examined the effects of dosing the WR-2721 by the i.v. route while continuing with the i.p. administration of the ormaplatin. WR-2721 was administered either 30 or 5 min before the ormaplatin, and the plasma and tissues were harvested at 15, 30, or 60 min after ormaplatin administration. The reverse-phase HPLC peak, which behaved chromatographically as a Pt(dach)(WR-1065) standard, was less prominent after the i.v. administration of WR-2721 than it was after i.p. administration under any of the paradigms tested. There was again no evidence for selectivity between tumor and nontumor tissue in the findings from any of the paradigms. It is concluded that if WR-2721 is capable of selectively protecting nontumor tissue from the toxicities of platinum-based chemotherapy, it is doing so by some mechanism other than its selective uptake into normal tissue and subsequent nonspecific inactivation of any reactive cytosolic platinum species formed. Other possible mechanisms are briefly discussed.

o-Methoxy-4-alkylphenols that form quinone methides of intermediate reactivity are the most toxic in rat liver slices.

The effects of p-alkyl substituents on the relative cytotoxicity of 4-alkyl-2-methoxyphenols were investigated in isolated rat liver slices. The derivatives of 4-alkyl-2-methoxyphenol studied were 4-methyl- (creosol), 4-ethyl-, 4-propyl-, 4-isopropyl-, 4-allyl-2-methoxyphenol (eugenol), as well as 4-allyl-2,6-dimethoxyphenol. The data were correlated with previous microsomal experiments which showed that all of the 4-alkyl-2-methoxyphenols were converted to quinone methides (QMs; 4-methylene-2,5-cyclohexadien-1-ones) via a cytochrome P450-catalyzed process [Bolton, J. L. Comeau, E., and Vukomanovic, V. (1995) Chem.-Biol. Interact., in press]. The present investigation showed little correlation between the rate of QM formation in microsomes and the relative toxicities of the alkylphenols, unless the QMs formed were of similar reactivity. In contrast, a plot of alkylphenol toxicity versus the relative hydrolysis rates of QMs derived from these phenols fit a parabolic equation with a minimum at the data for 4-isopropyl-2-methoxyphenol. These data suggest that in vivo oxidation of phenols to QMs which have lifetimes in the 10 s-10 min range results in cytotoxicity. QMs with reactivities outside this window are less toxic since the electrophile is either too stable for reaction with cellular nucleophiles or too reactive for nucleophilic cellular macromolecules to compete with solvent. These data suggest that a reactivity window exists for QMs which is a primary determinant of the extent of cytotoxic injury caused by these reactive electrophiles.

Inhibition of mitochondrial respiration by a para-quinone methide.

A relatively stable para quinone methide was prepared from 4-allyl-2,6-dimethoxyphenol. In aqueous solution the quinone methide had a half-life of 52 min, yet reacted rapidly with thiols such as glutathione or cysteine. The unusual stability of this quinone methide allowed us to directly test its effects on mitochondrial respiration. The quinone methide was a potent inhibitor of succinate-dependent mitochondrial respiration (IC50 = 47 microM). The inhibition appeared to be due to the depletion of protein thiols, since its effects were comparable to 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent). This quinone methide may prove a useful tool in the investigation of the specific effects of quinone methides on cells which lead to cytotoxicity.

Organ-specific biotransformation of ormaplatin in the Fischer 344 rat.

We examined the intracellular biotransformation products of ormaplatin [(d,l-trans)1,2-diaminocyclohexanetetrachloroplatinum(IV)] (formerly called tetraplatin) in liver, kidney, spleen, small intestine, and plasma of the adult male Fischer 344 rat. Previous studies have established that the rank order of ormaplatin toxicity in Fischer 344 rats is spleen approximately gastrointestinal tract > kidney >> liver. Animals were given tritium-labelled drug i.v. at 12.5 mg/kg, and tissues were harvested 30 min later. The kidney was found to concentrate total and cytosolic platinum to a greater extent than any of the other tissues. The absolute amount of cytosolic platinum, in micrograms per gram tissue, that was irreversibly bound to protein and/or other macromolecules was also greatest in the kidney. However, when the amount bound was expressed as a percentage of the total cytosolic platinum, the kidney was significantly lower than any other tissue. Of the various low molecular mass platinum biotransformation species characterized, by far the most abundant were complexes of platinum with the sulfur-containing molecules cysteine, methionine, and glutathione (GSH). There was more of the methionine complex in the blood plasma than in any of the tissues except for the spleen. No significant differences among the tissues were detected for the dichloro, cysteine, methionine, or the GSH complexes. The tritium-labelled diaminocyclohexane (DACH) carrier ligand appeared to remain stably bound to the platinum while in the plasma, as there was less free DACH ligand detected in plasma ultrafiltrate than in any tissue ultrafiltrate. Among the tissues, the free DACH levels were in the range of 20% of the radioactivity recovered from the HPLC column and were not significantly different. Consequently, neither biodistribution nor tissue-specific biotransformation of ormaplatin provides a ready explanation for the tissue specificity of ormaplatin toxicity in Fischer 344 rats. However, in kidney there was much less of the reactive PtCl2(DACH) species than has previously been reported for the corresponding Pt(NH3)2Cl2 species in cisplatin-treated rats. Thus, these data suggest a possible explanation for differences in nephrotoxicity induced by cisplatin versus that by ormaplatin.

The validity of self-reported smoking: a review and meta-analysis.

OBJECTIVES: The purpose of this study was to identify circumstances in which biochemical assessments of smoking produce systematically higher or lower estimates of smoking than self-reports. A secondary aim was to evaluate different statistical approaches to analyzing variation in validity estimates. METHODS: Literature searches and personal inquiries identified 26 published reports containing 51 comparisons between self-reported behavior and biochemical measures. The sensitivity and specificity of self-reports of smoking were calculated for each study as measures of accuracy. RESULTS: Sensitivity ranged from 6% to 100% (mean = 87.5%), and specificity ranged from 33% to 100% (mean = 89.2%). Interviewer-administered questionnaires, observational studies, reports by adults, and biochemical validation with cotinine plasma were associated with higher estimates of sensitivity and specificity. CONCLUSIONS: Self-reports of smoking are accurate in most studies. To improve accuracy, biochemical assessment, preferably with cotinine plasma, should be considered in intervention studies and student populations.