Biomedical subjects

Harish C Sikka

Publications and source records attributed to Harish C Sikka.

8 recordsLinked to original sources

PubMed2005

Attenuation of BPDE-induced p53 accumulation by TPA is associated with a decrease in stability and phosphorylation of p53 and downregulation of NFkappaB activation: role of p38 MAP kinase.

DNA damage caused by benzo[a]pyrene (B[a]P) or other polynuclear hydrocarbons (PAHs) induce p53 protein as a protective measure to eliminate the possibility of mutagenic fixation of the DNA damage. 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits p53 response induced by B[a]P and other DNA-damaging agents and may cause tumor promotion. The molecular mechanism of attenuation of B[a]P-induced p53 response by TPA is not known. We investigated the effect of TPA on p53 response in (+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)-treated mouse epidermal JB6(P(+)) Cl 41 cells. BPDE treatment induced p53 accumulation which was attenuated significantly by TPA. Cells treated with BPDE and TPA showed increased ratio of Mdm2 to p53 proteins in p53 immunoprecipitate and decreased p53 life span compared to BPDE-treated cells indicating p53 destabilization by TPA. TPA also inhibited BPDE-induced p53 phosphorylation at serine15. Activation of both ERKs and p38 MAPK by BPDE and attenuation of BPDE-induced p53 accumulation by U0126 or SB202190, specific inhibitor of MEK1/2 or p38 MAPK, indicate the role of ERKs and p38 MAPK in p53 accumulation. Interestingly, TPA potentiated BPDE-induced activation of ERKs whereas p38 MAPK activation was significantly inhibited by TPA, suggesting that inhibition of p38 MAPK is involved in p53 attenuation by TPA. Furthermore, SB202190 treatment caused decreased p53 stability and inhibition of phosphorylation of p53 at serine15 in BPDE-treated cells. We also observed that TPA or SB202190 attenuated BPDE-induced nuclear factor kappa B (NFkappaB) activation in JB6 Cl 41 cells harboring NFkappaB reporter plasmid. To our knowledge this is the first report that TPA inhibits chemical carcinogen-induced NFkappaB activation. Interference of TPA with BPDE-induced NFkappaB activation implicates abrogation of p53 function which has been discussed. Overall, our data suggest that abrogation of BPDE-induced p53 response and of NFkappaB activation by TPA is mediated by impairment of the signaling pathway involving p38 MAPK.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed2004

Mutagenicity of benzo[b]phenanthro[2,3-d]thiophene (BPT) and its metabolites in TA100 and base-specific tester strains (TA7001-TA7006) of Salmonella typhimurium: evidence of multiple pathways for the bioactivation of BPT.

Benzo[b]phenanthro[2,3-d]thiophene (BPT), and a number of its metabolites, including BPT-3,4-diol, BPT sulfoxide, BPT sulfone, and 3-hydroxyBPT were assessed for their mutagenic activity in Salmonella typhimurium strain TA100, and S. typhimurium base-specific strains TA7001, TA7002, TA7003, TA7004, TA7005, and TA7006. Among the compounds tested in strain TA100, BPT, BPT sulfone, and 3-hydroxyBPT did not show any significant mutagenic response in the presence of S9. In contrast BPT sulfoxide and BPT-3,4-diol (a precursor to the bay-region diol epoxide of BPT) showed significant mutagenic activity in the presence of S9. Surprisingly, BPT sulfoxide was nearly 3.3-fold more mutagenic than BPT-3,4-diol in the presence of S9. BPT sulfoxide also displayed intrinsic mutagenic activity, which was nearly 1.5-fold less than that displayed by BPT-3,4-diol in the presence of S9. In base specific tester strains, BPT sulfoxide was the most active metabolite in strains TA7002, TA7004, and TA7005 with S9 activation. In these strains, BPT-3,4-diol was 2- to 7-fold less mutagenic than BPT sulfoxide in the presence of S9. Only in strain TA7006, BPT-3,4-diol was four-fold more mutagenic than BPT sulfoxide. The fact that BPT sulfoxide is significantly more mutagenic than BPT-3,4-diol in S. typhimurium strain TA100 suggests that the formation of sulfoxide may be the principal pathway for the metabolic activation of BPT to mutagenic products. Based on the results from Tester Strain TA7005, it indicate that BPT and its most mutagenic metabolite BPT sulfoxide induce predominantly CG --> AT transversion, which is observed as the most frequent base substitution mutation of p53 tumor-suppressor gene in human lung cancer.

Animals↗

PubMed2004

Effects of cadmium(II) on (+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide-induced DNA damage response in human fibroblasts and DNA repair: a possible mechanism of cadmium's cogenotoxicity.

Cadmium, a widespread environmental pollutant and a cigarette smoke constituent, enhances the genotoxicity of benzo[a]pyrene (BP). The mechanism(s) underlying the potentiation of BP-induced genotoxicity by Cd2+ is not clearly understood. Our studies of the effects of noncytotoxic concentrations of Cd2+ on the levels of p53 and p21 in (+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)-treated human fibroblasts showed that Cd2+ decreased BPDE-induced p21 levels in a dose-dependent manner whereas p53 accumulation is attenuated only at higher noncytotoxic concentrations of cadmium. These findings suggest that both the activity and the accumulation of p53 in response of BPDE treatment are inhibited by Cd2+ although the possibility of p53-independent p21 transactivation cannot be ruled out. Exposure of synchronized human fibroblast cells to 0.5 microM of BPDE caused 72% of the cells remaining in G1 phase as compared to 52% in the case of untreated cells. Treatment of the cells with CdCl2 prior to exposing them to BPDE caused a decrease in the G1 population (72 to 54%) in a dose-dependent manner. An in vitro repair assay of BPDE-damaged pUC18 plasmid DNA using untreated and cadmium-treated nucleotide excision repair (NER) proficient HeLa extract showed that cadmium impaired the ability of HeLa cell extract to repair BPDE-damaged pUC18 DNA. Our findings indicate that cadmium not only inhibits NER pathway-dependent repair of BPDE-damaged DNA but also impairs p53 and p21 responses and overrides BPDE-induced G1-S cell cycle arrest. The effect of cadmium on these processes may explain, at least partly, the potentiating effect of the metal on the genotoxicity of BP.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed2004

Comparative metabolism of the aza polynuclear aromatic hydrocarbon dibenz[a,h]acridine by recombinant human and rat cytochrome P450s.

To assess the role of human and rat cytochrome P450s in the metabolism of aza-polynuclear aromatic hydrocarbons (aza-PAHs) and to examine the influence of heterocyclic nitrogen on the metabolism of these chemicals, we have investigated the biotransformation of dibenz[a,h]acridine (DB[a,h]ACR), an aza-PAH with two nonidentical bay regions, by recombinant human cytochromes P450 1A1, 1B1, and 3A4 and rat P450 1A1. Among the three P450s, 1A1 was the most effective in metabolizing DB[a,h]ACR followed by 1B1 and 3A4. The major DB[a,h]ACR metabolites produced by human P450 1A1 and 1B1 were the dihydrodiols with a bay region double bond, namely, DB[a,h]ACR-3,4-diol and DB[a,h]ACR-10,11-diol (putative proximate carcinogen). P450 1A1 produced a higher proportion of DB[a,h]ACR-10,11-diol (derived from the benzo ring adjacent to the nitrogen) (44.7%) than of DB[a,h]ACR-3,4-diol (derived from benzo ring away from the nitrogen) (23.8%). In contrast, 1B1 produced a much greater proportion of 3,4-diol (54.7%) than of 10,11-diol (6.4%). These data indicate that (i) human P450 1A1 and 1B1 differ dramatically with respect to the regiospecific metabolism of DB[a,h]ACR, (ii) human P450 1A1 is substantially more active than human P450 1B1 in the metabolic activation of the aza-PAH to its 10,11-diol, and (iii) the presence of nitrogen influences the relative extent to which the two benzo ring diols with a bay region double bond are formed by human P450s 1A1 and 1B1. In contrast to human P450s 1A1 and 1B1, rat P450 1A1 showed no regioselectivity in the metabolism of DB[a,h]ACR producing nearly equal proportions of 10,11-diol and 3,4-diol. Despite significant differences in their regioselectivity, human P450 1A1 and 1B1 and rat P450 1A1 showed similar stereoselectivity in the metabolism of DB[a,h]ACR to its diols having a bay region double bond, producing primarily the R,R enantiomers (>94%). The data of these studies indicate that human and rat P450 1A1 differ in their regioselectivity in the metabolism of DB[a,h]ACR to its two benzo ring diols with a bay region double bond and consequently in their ability to metabolically activate the parent aza-PAH. However, human and rat P450 1A1 do not differ with respect to their stereoselectivity in the metabolism of DB[a,h]ACR to the diols.

Acridines↗

PubMed2003

Metabolism of phenanthrene by brown bullhead liver microsomes.

We have investigated the regio- and stereoselective metabolism of phenanthrene by the liver microsomes of brown bullhead (Ameriurus nebulosus), a bottom dwelling fish species. The liver microsomes from untreated and 3-methylcholanthrene (3-MC)-treated brown bullheads metabolized phenanthrene at a rate of 14.1 and 20.7 pmol/mg protein/min, respectively, indicating that the hydrocarbon is a rather poor substrate for bullhead liver microsomes contrary to what has been reported for rat liver microsomes. The major phenanthrene metabolites formed by liver microsomes from untreated and 3-MC-treated bullheads included benzo-ring 1,2-dihydrodiol (25.3 and 11.6%), K-region 9,10-dihydrodiol (9.6 and 9.6%), and phenols (40.5 and 54.5%). The 3,4-dihydrodiol represented a minor proportion of the total phenanthrene metabolites. The low proportion of the 9,10-dihydrodiol formed by both control and 3-MC-treated bullhead microsomes sharply contrasts the previous data reported for the corresponding rat liver microsomes which metabolized phenanthrene predominantly to its 9,10-dihydrodiol representing 76.6 and 67.1%, respectively of the total metabolites. Liver microsomes from 3-MC-treated bullheads, like rat liver microsomes, were more selective in their attack at the 1,2-position of the benzo-ring than at the 3,4-position of the benzo-ring. Phenanthrene 1,2-dihydrodiol and 3,4-dihydrodiol formed by liver microsomes from both control and 3-MC-treated bullheads consisted predominantly of their R,R enantiomer. Phenanthrene, compared with benzo[a]pyrene and chrysene, is metabolized by bullhead liver microsomal enzymes to its benzo-ring dihydrodiols with a relatively low degree of stereoselectivity.

Animals↗

PubMed2003

Comparative metabolism of chrysene and 5-methylchrysene by rat and rainbow trout liver microsomes.

We have investigated the metabolism of chrysene (CHR) and 5-methychyrsene (5-MeCHR) by Shasta rainbow trout (Oncorhyncus mykiss) and Long Evans rat liver microsomes to assess the effect of a non-benzo ring methyl substituent on the reactions involved in the metabolism of polycyclic aromatic hydrocarbons (PAHs). Trout as well as rat liver microsomes metabolized both CHR and 5-MeCHR at essentially similar rates, indicating that the methyl substituent does not alter the substrate specificity of the cytochrome P450(s) involved in the metabolism of the two PAHs. Dihydrodiols were the major CHR metabolites formed by both trout and rat liver microsomes, whereas the trout liver microsomes formed a considerably higher proportion of 5-MeCHR phenols compared to diols, indicating that 5-methyl substitution alters the substrate specificity of trout microsomal epoxide hydrolase for 5-MeCHR epoxides. Unlike trout liver microsomes, rat liver microsomes formed a much greater proportion of 5-MeCHR diols compared to 5-MeCHR phenols, suggesting that 5-MeCHR epoxides are better substrates for the microsomal epoxide hydrolase present in rat liver than for the enzyme in trout liver. Both trout and rat liver microsomes are more efficient at attacking the bay-region bond versus the non-bay-region double bond in chrysene. In contrast the reverse is true in the case of 5-MeCHR, indicating that a non-benzo ring methyl substituent alters the regioselectivity of the enzymes involved in the oxidative metabolism of PAHs.

Animals↗

PubMed2003

Metabolism of the polynuclear sulfur heterocycle benzo[b]phenanthro[2,3-d]thiophene by rodent liver microsomes: evidence for multiple pathways in the bioactivation of benzo[b]phenanthro[2,3-d]thiophene.

Benzo[b]phenanthro[2,3-d]thiophene (BPT), a thia analogue of dibenz[a,h]anthracene (DBA), is a carcinogenic environmental pollutant. We have examined the metabolism of BPT by rodent liver microsomes to investigate the mechanism by which BPT produces mutagenic and carcinogenic effects. Both rat and mouse liver microsomes biotransformed [G-(3)H]BPT to various metabolites including BPT 3,4-diol and BPT sulfoxide, which are significantly more mutagenic than the parent compound. Liver microsomes from both control mice and rats metabolize BPT at similar rates. Treatment of mice with P450 inducers DBA, 3-methylcholanthrene (3-MC), Aroclor 1254, and phenobarbital enhanced the rate of metabolism of BPT by 74-, 28-, 77-, and 6-fold, respectively. In comparison, the treatment of rats with DBA and 3-MC increased the rate of metabolism of BPT by 22- and 34-fold, respectively, suggesting that P450 enzymes responsible for the metabolism of BPT are enhanced to different extents in rats and mice by a similar class of compounds. In general, the liver microsomes from mice treated with DBA or 3-MC were more active than those from similarly treated rats in metabolizing BPT to its 3,4-diol, a precursor to the bay-region diol epoxide of BPT. BPT sulfone was a minor metabolite (if formed) in all cases. The liver microsomes from rats treated with DBA or 3-MC or from mice treated with PB produced a significant proportion of BPT sulfoxide (12-41%). In contrast, the liver microsomes from DBA- or 3-MC-treated mice formed BPT sulfoxide as a minor metabolite (<2%). These studies indicate that cytochrome P450 enzymes induced by PAHs (e.g., P450 1A1 and P450 1B1) and by PB (e.g., P450 2B1, 3A1, and/or 3A2) are involved in the metabolism of BPT to mutagenic BPT 3,4-diol and BPT sulfoxide, providing evidence for the first time that BPT and possibly other thia-PAHs are metabolically activated via the formation of both the dihydrodiol (and subsequently diol epoxide) and the sulfoxide.

Animals↗

PubMed2003

Metabolism of chrysene by brown bullhead liver microsomes.

We have investigated the regio- and stereoselective metabolism of chrysene, a four-ring symmetrical carcinogenic polycyclic aromatic hydrocarbon (PAH), by the liver microsomes of brown bullhead (Ameriurus nebulosus), a bottom-dwelling fish species. The liver microsomes from untreated and 3-methylcholanthrene (3-MC)-treated brown bullheads metabolized chrysene at the rate of 30.1 and 82.2 pmol/mg protein/min, respectively. Benzo-ring diols (1,2-diol and 3,4-diol) were the major chrysene metabolites formed by liver microsomes from control and 3-MC-treated fish. However, the control microsomes produced a considerably higher proportion of chrysene 1,2-diol (benzo-ring diol with a bay region double bond) plus 1-hydroxychrysene, than 3,4-diol plus 3-hydroxychrysene, indicating that these microsomes are selective in attacking the 1,2- position of the benzo-ring. On the other hand, 3-MC-induced microsomes did not show such a regioselectivity in the metabolism of chrysene. Control bullhead liver microsomes, compared to control rat liver microsomes, produced a considerably higher proportion of chrysene 1,2-diol, the putative proximate carcinogenic metabolite of chrysene. Like rat liver microsomes, bullhead liver microsomes produced only trace amounts of the K-region diol. Chrysene 1,2-diol and 3,4-diol formed by the liver microsomes from both control and 3-MC-treated bullheads consisted predominantly of their R,R-enantiomers. Chrysene is metabolized by bullhead liver microsomal enzymes to its benzo-ring diols with a relatively lower degree of stereoselectivity compared to benzo[a]pyrene (a five-ring PAH), but with a higher degree of stereoselectivity compared to phenanthrene (a three-ring PAH). The data of this study, together with those from our previous studies with phenanthrene, benzo[a]pyrene and dibenzo[a,l]pyrene (a six-ring PAH), indicate that the regioselectivity in the metabolism of PAHs by brown bullhead and rainbow trout liver microsomes does not vary greatly with the size and shape of the molecule, whereas the degree of stereoselectivity in the metabolism of PAHs to benzo-ring dihydrodiols does.

Animals↗