Tzuu Huei Ueng

Acute and Subacute Toxicity Study of Water-Soluble Polyalkylsulfonated C60 in Rats

Polyalkylsulfonated C60, or FC4S, a highly water-soluble caged fullerene derivative, is believed to be a free radical remover or an antioxidant in biological systems. A 50 mg/ml aqueous solution was prepared as a master solution and administered to female Sprague-Dawley CD(Crl:CDR (SD)BR) rats in a single-dose acute toxicity study or a 12-day subacute toxicity study where rats were given the solution daily. In a study of the median lethal dose (LD50), no rats died after oral administration, and thus FC4S was considered to nontoxic if administered orally. In an LD50 intraperitoneal injection study, rats died within 30 hr after injection; the LD50 was determined to be approximately 600 mg per kilogram of body weight. Rats injected with the compound intraperitoneally or intravenously immediately eliminated the compound through the kidney; the kidney appeared to be the primary target organ. The compound induced a distinct lysosome-overload nephrosis, a phagolysosomal nephropathy characterized by a tinctorial difference between the outer cortex and the inner cortex and the medulla. The affected outer cortex showed a diffuse degeneration, with the presence of numerous large vacuoles and cytoplasmic aggregates in the tubular epithelium. The phagolysosomal nephropathy was detected in rats after acute exposure as well as in the surviving rats following 1 intraperitoneal injection of 500 mg/kg or intravenous injection of 100 mg/kg. Ultrastructural investigation revealed numerous membranous conglomerates characteristic of phagolysosomal and/or lysosomal inclusions in the cytoplasm of the renal tubular epithelium. These conglomerates were confined to the vacuole, electron-dense, and unevenly stained. They varied in size and shape and were fused or aggregated. Occasional phagolysosomes were also observed in the endothelial cells of the peritubular plexus. A preliminary study of microsomal enzyme activity analysis revealed a suppression effect of liver cytochrome P-450–dependent monooxygenase activities, including cytochrome P-450, cytochrome b5, and benzo(a)pyrene hydroxylase, but an increased level of kidney cytochrome P-450–dependent monooxygenase activities, including NADPH-cytochrome P-450 reductase. The significance of these enzyme alterations was not well determined. Further study is needed to clarify the correlation between the alterations of microsomal enzyme activity and the nephropathy of lysosomal overload-induced changes. These changes may serve as a biological marker in toxicity screening tests for this class of compound.

Suppression of microsomal cytochrome P450-dependent monooxygenases and mitochondrial oxidative phosphorylation by fullerenol, a polyhydroxylated fullerene C60.

The acute toxicity of fullerenol-1 was determined using mice pretreated intraperitoneally (i.p.) with polyhydroxylated C60 derivatives. The LD50 value of fullerenol-1 was estimated to be 1.2 g/kg. Pretreatments with 0.5 and 1.0 g/kg fullerenol-1 decreased cytochromes P450 and b5 contents, and NADPH-cytochrome P450 reductase, benzo[a]pyrene hydroxylase, 7-ethoxycoumarin O-deethylase, aniline hydroxylase, and erythromycin N-demethylase activities in liver microsomes. Pretreatments with 0.01 and 0.1 g/kg fullerenol-1 had no effect on these monooxygenases. Additions of fullerenol-1 to mouse liver microsomes suppressed monooxygenases activities toward benzo[a]pyrene, 7-ethoxycoumarin, aniline, and erythromycin with IC50 values of 42, 94, 102 and 349 microM, respectively. Fullerenol-1 exhibited noncompetitive and mixed-type of inhibition in benzo[a]pyrene hydroxylation and 7-ethoxycoumarin O-deethylation, respectively. Additions of fullerenol-1 to rat liver mitochondria resulted in a dose-dependent inhibition of ADP-induced uncoupling and markedly inhibited mitochondrial Mg2+ -ATPase activity with an IC50 value of 7.1 microM. These results demonstrate that fullerenol-1 can suppress the levels of the microsomal enzymes in vivo and decrease the activities of P450-dependent monooxygenase and mitochondrial oxidative phosphorylation in vitro.

INDUCTION OF CYTOCHROME P-450 1A1 IN HUMAN HEPATOMA HepG2 AND LUNG CARCINOMA NCI-H322 CELLS BY MOTORCYCLE EXHAUST PARTICULATE

The effects of motorcycle exhaust particulate (MEP) on human cytochrome P-450 (P-450)-dependent monooxygenases were determined using human hepatoma cell line HepG2 and lung carcinoma cell line NCI-H322 treated with organic extracts of MEP from a two-stroke engine. Gas chromatography and mass spectrometry analysis of MEP extract revealed the presence of carcinogens benzo[a]pyrene, benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[g,h,i]perylene, chrysene, and indeno[1,2,3-c,d]pyrene in the chemical mixture. Treatment with MEP extract produced concentration- and time-dependent increases of monooxygenase activity in HepG2 cells. Treatment of the cells with 100 µg/ml MEP extract for 24 h markedly increased benzo[a]pyrene hydroxylation, 7-ethoxycoumarin, and 7-ethoxyresorufin O-deethylation activities in microsomes. Immunoblot analysis of microsomal proteins using mouse monoclonal antibody 1-12-3 against P-450 1A1 revealed that MEP extract induced a P-450-immunorelated protein in the hepatoma cells. RNA blot analysis of cellular total RNA using a human P-450 1A1 3?-end cDNA probe showed that MEP extract increased the level of a hybridizable P-450 mRNA. These P-450 1A1 inductive effects of MEP extract were similar to those from treatment with 10 µM benzo[a]pyrene or 3-methylcholanthrene (3-MC) in HepG2 cells. Treatment of lung carcinoma NCI-H322 cells with 100 µg/ml MEP extract, 10 µM benzo[a]pyrene, or 3-MC resulted in induction of monooxygenase activity, protein, and mRNA of P-450 1A1, similar to the induction observed with the hepatoma cells. The present study demonstrates that MEP extract has the ability to induce human hepatic and pulmonary P-450 1A1 in the liver- and lung-derived cell lines, and the induction involves a pretranslational mechanism. Induction of the human hepatic and pulmonary P-450 1A1 in vitro may provide important information in the assessment of MEP metabolism and toxicity in humans.

Modulation of cytochrome P-450-dependent monooxygenases, glutathione and glutathione S-transferase in rat liver by geniposide from Gardenia jasminoides

Geniposide is an iridoid glycoside extracted from the fruits of Gardenia jasminoides, which are used as a food colorant and as a traditional Chinese medicine for treatment of hepatic and inflammatory diseases. The effects of geniposide and G. jasminoides fruit crude extract on liver cytochrome P-450 (P-450)-dependent monooxygenases, glutathione and glutathione S-transferase were investigated using rats treated orally with the iridoid glycoside (0.1 g/kg body weight/day) or the fruit crude extract (2 g/kg/day) for 4 days. The treatments decreased serum urea nitrogen level but increased liver to body weight ratio, total hepatic glutathione content and hepatic cytosolic glutathione S-transferase activity. Treatments with geniposide and G. jasminoides decreased P-450 content, benzo[a]pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, and erythromycin N-demethylation activities in liver microsomes without affecting aniline hydroxylation activity. The natural products had no effect on glutathione content and monooxygenase activities in kidney microsomes. Immunoblotting analyses of liver microsomal proteins using mouse monoclonal antibody 2-13-1 to rat P4503A1/2 revealed that geniposide and G. jasminoides crude extract decreased the intensity of a P4503A-immunorelated protein. Protein blots probed with mouse monoclonal antibody 1-12-3 to rat P4501A1 and rabbit polyclonal antibody against human P4502E1 showed that both treatments had little or no effect on P4501A and 2E proteins. The present findings demonstrate that geniposide from G. jasminoides has the ability to inhibit a P4503A monooxygenase and increase glutathione content in rat liver.

Induction of cytochrome P450 1A in hamster liver and lung by 6-nitrochrysene

Abstract The present study has determined the effect of 6-nitrochrysene (6-NC) on hepatic and pulmonary cytochrome P450 (P450)-dependent monooxygenases using hamsters pretreated with the nitrated polycyclic aromatic hydrocarbon (nitro-PAH) at 5 mg/kg per day for 3 days. Pretreatment with 6-NC elevated serum gamma-glutamyltranspeptidase, lactate dehydrogenase, and bilirubin levels. Liver S9 fractions prepared from controls and hamsters pretreated with 6-NC markedly increased mutagenicity of the nitro-PAH in Salmonella typhimurium tester strains TA98, TA100, and TA102. The pretreatment selectively increased 1-nitropyrene reductase activities of lung cytosol and liver and lung microsomes. Pretreatment with 6-NC resulted in increases of microsomal 7-ethoxyresorufin and methoxyresorufin O-dealkylases activities in liver and lung without affecting the monooxygenase activities in kidney. Immunoblot analysis of microsomal proteins using mouse monoclonal antibody 1–12–3 to rat P450 1A1 revealed that 6-NC induced P450 1A-immunorelated proteins in liver and lung. RNA blot analysis using mouse P450 1A1 cDNA showed that 6-NC increased liver and lung P450 1A mRNA. 6-NC had no effect on the kidney P450 protein and mRNA. The present study demonstrates that the hamster enzymes can support 6-NC metabolic activation and the nitro-PAH induces liver and lung P4501A via a pretranslational mechanism.

Potential roles of fibroblast growth factor-9 in the benzo(a)pyrene-induced invasion in vitro and the metastasis of human lung adenocarcinoma

Fibroblast growth factor (FGF)-9 belongs to the FGF family which modulate cell proliferation, differentiation, and motility. Benzo(a)pyrene is a polycyclic aromatic hydrocarbon (PAH) and ubiquitous environmental carcinogen present in automobile exhaust, cigarette smoke, and foods. The major purposes of this study were to explore the roles of FGF-9 in the benzo(a)pyrene-induced lung cancer invasion in vitro and the metastatic development of lung adenocarcinoma in human. The data of RT-PCR analysis indicated that treatments of human lung adenocarcinoma CL5 cells with benzo(a)pyrene and a PAH mixture motorcycle exhaust particulate (MEP) extracts increased FGF-9 mRNA expression. The increased expression was blocked by cotreatments with a p38 mitogen-activated protein kinase inhibitor SB202190 and an extracellular signal-regulated kinase inhibitor PD98059. The results of immunoblot analysis and Matrigel assay showed that benzo(a)pyrene and MEP extracts produced a concomitant induction of FGF-9 protein and invasive ability of CL5 cells. The benzo(a)pyrene- and MEP-induced invasion was suppressed by FGF-9 neutralizing antibodies. The results of immunohistochemistry analysis of human lung adenocarcinoma specimens showed that FGF-9 protein was detected in the adenocarcinoma cells but not in normal epithelium. FGF-9 staining intensity was positively correlated with status of disease and degree of lymph node metastasis in these lung adenocarcinomas. These present findings suggest that FGF-9 has potential roles in benzo(a)pyrene-induced CL5 cell invasion and human lung adenocarcinoma metastasis.

Modulation of Microsomal Cytochrome P450 by Scutellariae Radix and Gentianae Scabrae Radix in Rat Liver

The present study has determined the effects of Scutellariae Radix (Huangqin) and Gentianae scabrae Radix (Longdan) on liver microsomal cytochrome P450 (P450)-dependent mono-oxygenases using rats pretreated with crude extracts of medicinal herbs. Scutellariae Radix resulted in a 53% decrease of pentoxyresorufin O-dealkylase activity in liver microsomes. In contrast, Gentianae scabrae Radix caused a 50% increase of benzo(a)pyrene hydroxylase activity. Immunoblotting analysis of liver microsomes revealed that Scutellariae Radix induced and suppressed the levels of P450 1A and 2B proteins, respectively. Scutellariae and Gentianae scabrae Radixes had no effects on microsomal aniline hydroxylase activity and P450 2E1 protein.

Induction of cytochromes P450 1A, 2B and 2E in hamster tissues by acetone

Abstract The inductive effects of acetone on cytochromes P450 1A, 2B and 2E in liver, kidney and lung were studied using hamsters administered acetone in drinking water. Acetone administration caused five-, two- and sixfold increases of the activities of aniline hydroxylase, ethoxyresorufin O-dealkylase and pentoxyresorufin O-dealkylase in liver microsomes; eight- and twofold increases of aniline hydroxylation and ethoxyresorufin O-dealkylation in kidney; and a twofold increase of aniline hydroxylation in lung, respectively. Immunoblot analyses of microsomal proteins using mouse monoclonal antibody 1-12-3 to rat P450 1A1 and rabbit antibody to human P450 2E1 revealed that acetone resulted in about three-, four- and twofold increases of proteins immunorelated to P450s 1A and 2E in hamster liver, kidney, and lung, respectively. Protein blot analysis using mouse monoclonal antibody 2-66-3 to rat P450 2B1 showed that acetone caused five- and twofold increases, respectively, of an immunoreactive protein in hamster liver and kidney but decreased the P450 2B protein by 48% in lung. RNA blot analyses using cDNA probes derived from mouse P450 1A1 and rat P450 2B1 cDNA clones demonstrated that acetone elicited two- and twelvefold increases of the mRNA levels of P450s 1A and 2B in hamster liver, respectively. Northern blot analyses using oligonucleotide probes derived from hamster P450 2E1 cDNA sequence detected two species of hybridizable mRNA in control liver. Acetone preferentially caused a threefold increase in the level of the larger-sized mRNA. Acetone produced little increase or no effect on mRNAs of cytochromes P450 1A, 2B and 2E in kidney and lung. The present study demonstrates that acetone induces the catalytic activity, protein and mRNA levels of P450s 1A, 2B and 2E in hamster liver, and causes various changes of the P450 levels in kidney and lung. Acetone induction of hamster P450s 1A, 2B and 2E might involve transcriptional and post-transcriptional mechanisms.