Proteomic profiles of induced hepatotoxicity at the subcellular level

In the present study proteomes of liver samples were analyzed after administration of phenobarbital (PB) or 3-methylcholantrene (3-MC) to mice. Liver cell homogenates were subfractionated by differential ultracentrifugation into cytosol and microsomes, which were subjected to 2-DE to generate the proteomic maps of these fractions. 2-DE yielded 1100 and 800 protein spots for microsomes and cytosol, respectively. General trends of the fraction-specific alterations after 3-MC or PB treatment were evaluated using the Student's t-test and the principal component analysis (PCA). According to the PCA-derived data, the microsomal changes after 3-MC and PB treatment were quite similar. However, in the case of the cytosol data, the specificities of 3-MC- and PB-induced responses could be clearly distinguished from each other. Protein spots, whose expression levels differed from control, were identified by MALDI-TOF PMF. Proteomic studies such as those reported herein can be useful in identifying the molecular-based toxicity of lead drug candidates.     

Fungal oxidation of 3-methylcholanthrene: Formation of proximate carcinogenic metabolites of 3-methylcholanthrene

The filamentous fungus, Cunninghamella elegans, was found to metabolize the potent carcinogen, 3-methylcholanthrene (3-MC) to 1-hydroxy-3-MC, 2-hydroxy-3-MC, 1-keto-3-MC, 2-keto-3-MC and trans-9,10-dihydrodiols of 1-hydroxy-3-MC. In addition several unidentified derivatives of 3-MC were found. The metabolites formed were separated by high pressure liquid chromatography (HPLC) and identified by comparison of retention times, absorbance, fluorescence and mass spectra with those of synthetic standards. Incubation of (±)-1-hydroxy-3-MC and (±)-2-hydroxy-3-MC with cells of C. elegans indicated that 1-hydroxy-3-MC is metabolized to form diasteromerically related trans-9,10-dihydrodiols of 1-hydroxy-3-MC. Experiments with 3-[¹⁴C]MC showed that over a 48-h period, 8.7% of the hydrocarbon was oxidized to organic solvent-soluble metabolic products. Most of the metabolites were polar products, some of which co-chromatographed with trans-9,10-dihydrodiols of 1-hydroxy-3-MC. The results show that C. elegans has the ability to oxidize 3-MC to metabolites that have been implicated as proximate carcinogenic forms of 3-MC in higher organisms.     

The time course of effects of cadmium and 3-methylcholanthrene on activities of enzymes of xenobiotic metabolism and metallothionein levels in the plaice, Pleuronectes platessa

Plaice were treated with an acute dose of a polyaromatic hydrocarbon (3-methylcholanthrene, 3-MC) or cadmium, or 3-MC and cadmium by i.p. injection. The effects on hepatic detoxication systems, cytochrome P-450 (ethoxyresorufin O-deethylase, EROD), UDP-glucuronyl transferase, glutathione S-transferase, glutathione peroxidase activities, total glutathione (GSH), metallothionein and Cd and Zn in the cytosol were studied over a 14 day period. 3-MC increased EROD (7-18-fold), glucuronyl transferase (40%) and GSH transferase (200%) activities, whereas GSH peroxidase activity decreased by 60%. Cd treatment inhibited EROD (90%), GSH transferase (90%) and GSH peroxidase (30%) activities and displaced Zn. Total GSH levels increased (200%) prior to onset of metallothionein synthesis (6 days). Cotreatment with 3-MC and Cd led to a marked increase in GSH levels (300%) but the onset of metallothionein synthesis was delayed by a week. Induction of enzyme activities was abolished, EROD activity was strongly inhibited and there was a transient 50-90% decrease in glucuronyl transferase, GSH transferase and GSH peroxidase activities on days 2 and 3 after treatment. The results indicate that a polyaromatic hydrocarbon could result in increased peroxidative damage, the heavy metal Cd can severely inhibit organic xeno- and endobiotic metabolism and that the effects of both agents may be synergistic.     

The use of high pressure liquid chromatography to study chemically induced alterations in the pattern of benzo[a]pyrene metabolism.

The metabolism of radiolabeled benzo[a]pyrene (BP) by control, 3-methyl-cholanthrene (3-MC) induced, and 1,1,1-trichloropropene-2,3-oxide (TCPO)-inhibited rat liver microsomes was measured using fluorescence, radiometric, and high-pressure liquid chromatographic (HPLC) assays. Significant differences in the total measurable metabolism of BP by the three microsomal enzyme incubations resulted from the use of the three assay procedures. Appreciable differences in the concentration of the metabolite fractions after 3-MC induction and TCPO inhibition are clearly demonstrated. NMR analysis revealed that while the 3-hydroxy-BP fraction is greater than 90% pure, the 9-hydroxy fraction contains a number of metabolites having essentially identical retention times.     

Nucleolar activity after 3-methylcholanthrene treatment of rat liver cells studied by silver staining procedure

The influence of a single dose of 3-methylcholanthrene (3-MC) was studied in nucleoli of young rat liver cells by means of conventional and ultracytochemical methods. The nucleolar activity was stimulated in our experimental conditions: the appearance of the fibrillar centres in the liver cell nucleoli as well as the silver staining protein content of the fibrillar centres and the dense fibrillar component were increased by 3-MC. The results suggest that the activity of ribosomal genes was increased following 3-MC treatment.     

Effect of 3-methylcholanthrene on the biliary excretion of bromsulphthalein and eosine in newborn rats.

The biliary excretion rates of bromsulphthalein (BSP), bromsulphthalein-glutathione conjugate (BSP-GSH) and eosine have been studied in 3-methylcholanthrene (3-MC)-pretreated (100 mg/kg i.p.) and control rats aged 10 days. Liver weight was invariably increased after 3-MC treatment, associated with enhanced biliary excretion of total BSP. The increase in the biliary excretion of total BSP was due solely to the increased excretion of BSP-GSH. Following 3-MC pretreatment, BSP-GSH and eosine appeared in the bile in the same amount as in the control rats after i.v. administration of BSP-GSH and eosine. Pretreatment with 3-MC increased the ratio of BSP-GSH to BSP in the liver and bile. Our results suggest that the increased biliary excretion of total BSP following 3-MC treatment was due to an enhanced conjugation of BSP with GSH.     

The effect of inducing agents on the metabolism of ethidium bromide by isolated rat hepatocytes

The metabolism of ethidium bromide by isolated rat hepatocytes is significantly enhanced by pre-treatment of animals with phenobarbitone (PB) and 3-methylcholanthrene (3-MC). Pre-treatment with PB and 3-MC results in a 2.5- and 1.5-fold increase, respectively in the amount of the principal metabolite, ethidium 8-N-glucuronide, compared with that formed by hepatocytes from untreated rats. The formation of ethidium 3-N-glucuronide is not enhanced by pre-treatment with either PB or 3-MC. Two new metabolites, hydroxyethidium glucuronide and a transient unidentified species, have been detected by HPLC and are formed only by hepatocytes from animals pre-treated with 3-MC.     

Oltipraz inhibits 3-methylcholanthrene induction of CYP1A1 by CCAAT/enhancer-binding protein activation

Oltipraz, a cancer chemopreventive agent, induces CYP1A1 to a certain extent by transactivation of the gene via the Ah receptor (AhR)-xenobiotic response element (XRE) pathway. Previously, we showed that oltipraz promoted CCAAT/enhancer binding proteinbeta (C/EBPbeta) activation, which leads to the induction of glutathione S-transferase. Given that oltipraz activates C/EBPbeta for gene transactivation and that the putative C/EBP binding site is located in the CYP1A1 promoter region, this study investigated the effect of oltipraz on CYP1A1 induction by 3-methylcholanthrene (3-MC). 3-MC induced CYP1A1 in H4IIE cells in a time- and concentration-dependent manner. Gel shift analysis showed that 3-MC increased the band intensity of protein binding to the XRE. Immunocompetition analysis verified the specificity of AhR-XRE binding. Oltipraz (30 microM) induced CYP1A1 and the CYP1A1 promoter-luciferase gene and increased AhR DNA binding activity, which was 10-20% of those in 3-MC (100 nM)-treated cells. However, AhR-XRE binding was not increased after 10 microM oltipraz treatment. Oltipraz (10 microM) significantly inhibited CYP1A1 and CYP1A1-luciferase gene induction by 3-MC with no increase in AhR DNA binding. Oltipraz enhanced protein binding to the C/EBP binding site in the gene promoter and the binding complex comprised of C/EBPbeta and partly C/EBPdelta. Overexpression of dominant-negative mutant C/EBP significantly abolished the ability of oltipraz to suppress 3-MC-inducible CYP1A1 and the CYP1A1 reporter gene expression. Consistently, C/EBPbeta overexpression blocked CYP1A1 reporter gene induction by 3-MC. These results provide evidence that oltipraz suppresses 3-MC induction of CYP1A1 gene expression and that activation of C/EBPbeta by oltipraz contributes to suppression of 3-MC-inducible AhR-mediated CYP1A1 expression.     

The effect of Sudan III on drug metabolizing enzymes

We have examined the induction of drug metabolizing enzymes in rat liver microsomes by azo dye, 1-(p-phenylazophenylazo)-2-naphthol (Sudan III). Marked increases were observed in the levels of cytochrome P-448 as well as in p-nitroanisole O-demethylase (p-NAD), amaranth (AR) and neoprontosil reductases (NPR) and 7-ethoxycoumarin O-deethylase (ECD) activities. On the other hand, aminopyrene N-demethylase activity was not significantly increased. Further, induced ECD activity was inhibited 90% by a specific antibody against cytochrome P-448 while the inhibition observed with an antibody against cytochrome P-450 was less than 25%. Simultaneous administration of Sudan III and 3-methylcholanthene (3-MC) induced cytochrome P-448 up to a level brought about by either Sudan III or 3-MC treatment alone. In contrast, Sudan III did not induce cytochrome P-448 in the 3-MC insensitive DBA/2 mouse. Solubilized microsomes from Sudan III-treated rats showed an identical sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) pattern with those from 3-MC-treated animals. It is concluded that the cytochrome P-448 induced in liver by Sudan III is very similar to that induced by 3-MC. Sudan III also induced UDP-glucuronyltransferase activity towards 1-naphthol and estradiol. It did not induce NADPH-cytochrome c reductase, nor any of the enzymes which constitute the microsomal electron transport chain except for cytochrome P-448.     

Transformation of SV40-immortalized human uroepithelial cells by 3-methylcholanthrene increases IFN- and Large T Antigen-induced transcripts

Background  

Simian Virus 40 (SV40) immortalization followed by treatment of cells with 3-methylcholanthrene (3-MC) has been used to elicit tumors in athymic mice. 3-MC carcinogenesis has been thoroughly studied, however gene-level interactions between 3-MC and SV40 that could have produced the observed tumors have not been explored. The commercially-available human uroepithelial cell lines were either SV40-immortalized (HUC) or SV40-immortalized and then 3-MC-transformed (HUC-TC).     

Induction of DT-diaphorase by 2,3,7,8-tetrachlorodibenzo-p-dioxin (ICDD).

The compounds 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-$\text{p}$-dioxin (TCDD) are both inducers of the enzyme system aryl hydrocarbon hydroxylase. It has recently been reported that 3-MC is also an inducer of DT-diaphorase activity in rat liver. In this report the ability of TCDD to induce hepatic DT-diaphorase activity was examined. The results indicate that TCDD is approximately 17,000 times more potent as an inducer of DT-diaphorase activity than 3-MC.     

Scoparone O-demethylase assay is not useful to differentiate the effects of model inducers of cytochrome P-450 in rabbit and guinea pig liver

Coumarin derivative, scoparone (6,7-dimethoxycoumarin), is regioselectively O-demethylated into isoscopoletin (I) and scopoletin (S). This oxidation is inversely influenced by cytochrome P-450 inducers in the rat such as 3-methylcholantrene (3-MC) and phenobarbital (PB). The I/S ratio is higher than 1.5 with 3-MC treatment whereas it is lower than 0.5 with PB treatment. With regards to this contrasting effect, it gas been suggested that the I/S ratio should be useful to differemtiate between the effects of these types of inducers. We studied the consequences of in vivo PB and 3-MC treatment on scoparone biotransformation in guinea pig and rabbit. In these two species, at the basal state, scoparone biotransformation was enhanced in comparison to the rat. Moreover, in these untreated animals, two other metabolites were formed. After 3-MC or PB treatment, scoparone metabolism is, in contrast to the rat, inappropriate to differentiate between the P-450 profile of other animals.     

Effect of chronic treatment with phenobarbital or 3-methylcholanthrene on the male reproductive system in rats

Treatment of rats for 4 weeks with phenobarbital (PB) did not inhibit the growth of the seminal vesicles, nor did it affect the biosynthesis of testosterone by testis microsomes. Moreover, neither the concentration of cytochrome p-450 or the 17 α-hydroxylase activity in testis microsomes were affected. In contrast, treatment with 3-methylcholanthrene (3-MC) for 4 weeks markedly decreased the weights of the seminal vesicles. The decrease was probably related to an impairmant of testosterone formation in the gonads, since testosterone biosynthesis as well as the concentration of cytochrome p-450 and the activity of 17 α-hydroxylase in testis microsomes were significantly decreased in the 3-MC treated rats. No histopathological changes were seen in testes from any of the PB or 3-MC treated rats.     

A test using cultured cells with induced mixed-function oxygenase in the unscheduled DNA synthesis assay for detecting promutagens/procarcinogens

The human FL cell line contains very low levels of constitutive AHH activity, but it could be greatly induced by NE, beta-NF and 3-MC, and induced slightly by PB. When two different types of inducer, for example, 3-MC and PB or 3-MC and NE were given in combination, an additive inductive effect was not observed. Both the constitutive and induced AHH in FL cells have characteristics of MFO, namely, NADPH-dependence and CO-sensitivity. The fact that the constitutive and induced AHH in FL cells could be inhibited by a known hydroxylase inhibitor 7,8-BF indicated that the AHH in FL cells belongs to the cytochrome P-448 dependent MFO type. After removal of inducer from the medium, the induced AHH activity remained at a high level for at least 24-36 h. By using AHH-induced FL cells in the UDS assay system for the detection of promutagens/procarcinogens, we found that AFB1 and 3-MC did not induce a UDS reaction in uninduced FL cells, while in beta-NF induced cells, 10(-6)-10(-4) M AFB1 and 10(-7)-10(-6) M 3-MC elicited a very significant UDS reaction, which was concordant with the results obtained in the UDS assay system using HeLa cells or FL cells supplemented with liver microsomes or using primary cultured hepatocytes as indicator cells. B(a)P elicited the UDS reaction at concentrations of 10(-6)-10(-3) M in beta-NF induced cells, whereas 10(-4)-10(-3) M was required in uninduced cells. The results above indicate that this new design is feasible, but further study is needed to assure its accuracy.     

The effect of antithymocyte serum on the natural cytotoxicity of C3HA mouse splenocytes in the early periods after 20-methylcholanthrene administration]

The role of T-lymphocytes in natural cytotoxicity of splenocytes of C3HA mice after a single injection of 20-methylcholanthrene (20-MC) was investigated. A splenocyte suspension was treated with anti-T-cell serum and complement. This treatment was not shown to exert influence on the natural cytotoxicity of splenocytes within 1-13 days after injecting 20-MC.     

Intracerebroventricular 4-methylcatechol (4-MC) ameliorates chronic pain associated with depression-like behavior via induction of brain-derived neurotrophic factor (BDNF)

Neuropathic pain concurrent with mood disorder from peripheral nerve injury is a serious clinical problem that significantly affects quality of life. Recent studies have suggested that a lack of brain-derived neurotrophic factor (BDNF) in the limbic system may cause this pain-emotion. BDNF is induced in cultured neurons by 4-methylcatechol (4-MC), but the role of 4-MC-induced BDNF in pain-emotion is poorly understood. Thus, we assessed the possible involvement of BDNF in brain in depression-like behavior during chronic pain following peripheral nerve injury. In addition, we examined whether intracerebroventricular (i.c.v.) 4-MC prevents chronic pain in rats and produces an antidepressant effect. Sprague-Dawley rats implanted intracerebroventricularly with a PE-10 tube were subjected to chronic constriction injury (CCI). Pain was assessed by a reduction in paw withdrawal latency (PWL) to heat stimuli after CCI. We also used a forced swimming testing (FST; time of immobility, in seconds) from day 14 to day 21 after CCI. Modulation of pain and emotional behavior was performed by injection of PD0325901 (a MEK1/2 inhibitor). 4-MC (100 nM) was continuously administered i.c.v. for 3 days during the period from day 14 to day 21 after CCI. To block analgesic and antidepressant effects, anti-BDNF antibody or K252a (a TrkB receptor inhibitor) was injected in combination with 4-MC. Naloxone was also coadministered to confirm the analgesic effect of 4-MC. During the chronic stage after CCI, the rats showed a sustained decrease in PWL (thermal hyperalgesia) associated with extension of the time of immobility (depression-like behavior). PD0325901 significantly reduced the decrease in PWL and the increased time of immobility after CCI. The decreased PWL and increased time of immobility were also reduced by 4-MC and by treatment with an ERK1/2 inhibitor. These effects of 4-MC i.c.v. were reversed by anti-BDNF and K252a. The analgesic effect of 4-MC i.c.v. was also antagonized by naloxone. Based on these results, we suggest that a lack of BDNF and activation of ERK1/2 in the pain-emotion network in the CNS may be involved in depression-like behavior during chronic pain. 4-MC i.c.v. ameliorates chronic pain and depression-like behavior by producing of BDNF and normalization of ERK1/2 activation. Therefore, enhancement of BDNF may be a new treatment strategy for chronic pain associated with depression.     

Cutaneous metabolism of benzo[a]pyrene: comparative studies in C57BL/6N and DBA/2N mice and neonatal Sprague--Dawley rats

The metabolism of the polycyclic aromatic hydrocarbon (PAH) carcinogen benzo[a]pyrene (BaP) was studied using microsomes prepared from the skin of the mouse and rat. Topical application of the polychlorinated biphenyl (PCB) Aroclor 1254 or the PAH 3-methylcholanthrene (3-MC) to the skin of the C57BL/6N and DBA/2N mouse and the Sprague-Dawley rat caused statistically significant enhancement of cutaneous microsomal aryl hydrocarbon hydroxylase (AHH) activity in each animal. PCB was a more potent inducer of the enzyme than was 3-MC. BaP metabolism by skin microsomes from the same animals was assessed using high performance liquid chromatography (HPLC). The skin of untreated animals metabolized BaP into 9,10-, 7,8- and 4,5-dihydrodiols, phenols and quinones. Skin application of PCB caused greater than 16–18-fold enhancement of BaP metabolism in the C57BL/6N mouse and the rat and 2–5-fold enhancement in the DBA/2N mouse. Skin application of 3-MC enhanced BaP metabolism 2–8-fold in the C57BL/6N mouse and 5–10-fold in the rat and had no effect in the DBA/2N mouse. The formation of procarcinogenic metabolite BaP-7, 8-diol was greatly enhanced (4–12-fold) by treatment with the PCB and 3-MC in the tumor susceptible C57BL/6N mouse and in the tumor-resistant neonatal Sprague-Dawley rat. In contrast, the formation of BaP-7,8-diol was either slightly enhanced (2-fold) or unaffected by treatment with the PCB or 3-MC in the tumor-resistant DBA/2N mouse. Our data indicate that neither the patterns of metabolism nor the amount of BaP-7,8-diol formation in the skin are reliable predictors of tumor susceptibility to the PAH in rodent skin.     

Interaction of 18-methoxycoronaridine with nicotinic acetylcholine receptors in different conformational states

The interaction of 18-methoxycoronaridine (18-MC) with nicotinic acetylcholine receptors (AChRs) was compared with that for ibogaine and phencyclidine (PCP). The results established that 18-MC: (a) is more potent than ibogaine and PCP inhibiting (±)-epibatidine-induced AChR Ca²⁺ influx. The potency of 18-MC is increased after longer pre-incubation periods, which is in agreement with the enhancement of [³H]cytisine binding to resting but activatable Torpedo AChRs, (b) binds to a single site in the Torpedo AChR with high affinity and inhibits [³H]TCP binding to desensitized AChRs in a steric fashion, suggesting the existence of overlapping sites. This is supported by our docking results indicating that 18-MC interacts with a domain located between the serine (position 6′) and valine (position 13′) rings, and (c) inhibits [³H]TCP, [³H]ibogaine, and [³H]18-MC binding to desensitized AChRs with higher affinity compared to resting AChRs. This can be partially attributed to a slower dissociation rate from the desensitized AChR compared to that from the resting AChR. The enthalpic contribution is more important than the entropic contribution when 18-MC binds to the desensitized AChR compared to that for the resting AChR, and vice versa. Ibogaine analogs inhibit the AChR by interacting with a luminal domain that is shared with PCP, and by inducing desensitization.