Relative quantification of CYP1A gene expression in whitefish (Coregonus lavaretus) exposed to benzo[a]pyrene

The expression of CYP1A (cytochrome P4501A) can be induced by a number of aromatic compounds in teleost fishes. We developed a real-time PCR assay for measuring relative quantities (RQ) of CYP1A mRNA in whitefish (Coregonus lavaretus). To test for the usefulness of the assay we performed a treatment study, using benzo[a]pyrene (B[a]P) a model CYP1A inducer. Primers for the CYP1A gene were adapted from the literature, whereas those for [beta]-actin (endogenous control) were designed from a region that was found to be conserved among salmonid [beta]-actin genes. A group of hatchery raised whitefish, with an average body mass of 15 g and total length of 12 cm were given an intraperitoneal injection (10 mg/kg) of B[a]P in corn oil (2 mg B[a]P/ml corn oil) or corn oil alone (Control). After 48 h, whitefish liver, head kidney and brains were collected for mRNA isolation and analysis. In all three tissues sampled, CYP1A mRNA was affected by treatment with B[a]P. Head kidney tissue showed the greatest induction potential (RQ=11.00) from base levels (RQ=1.00), followed by liver (RQ=9.45), and brain (RQ=3.76). These results demonstrated that CYP1A was highly inducible by B[a]P in whitefish head kidney and liver, and to some extent, in brain tissue. The approach presented here has the advantage of providing rapid and accurate measures of CYP1A induction in various tissues of fish responding to PAH contaminant exposure

Benzo[a]pyrene and cyclopenta[c]phenanthrene suppress expression of p53 in head kidney of rainbow trout

Although p53, a protein of important tumor suppressive function, has been extensively studied in mammals, relatively little is known about the p53 pathways in lower vertebrates. Particularly, limited information exists on possible influences of environmental contaminants on the expression of the p53 gene in fish. In the current study, we assessed the effects of benzo[a]pyrene (B[a]P; potent tumor promoter) and cyclopenta[c]phenanthrene (CP[c]Ph; clastogenic agent) exposure on a 24h profile of p53 gene expression in head kidney of juvenile rainbow trout (Oncorhynchus mykiss). To analyze the p53 transcription rate, we developed protocol for the examination of both mRNA and heterogeneous nuclear (hn) RNA of the gene, using Real-Time RTPCR approach. The results show that both compounds are capable of suppressing p53 transcriptional activity within 12h of the treatment. Our finding supports the idea that structurally different PAHs may influence cell physiologic functions controlled by p53 in fish, in part, by down-regulating its RNA expression levels

Oxidative metabolism, mutagenic and carcinogenic properties of some polycyclic aromatic hydrocarbons

This attempt to synthesize the present state of knowledge on the correlation between the structure of chosen PAHs and their mutagenic or carcinogenic activity shows that the dependencies cannot be treated univocally. Extreme complexity of the subject, typical for investigations on the boundary between chemistry and biology, inclines one to search for new ways of synthesis of new compounds, which would be of interest for biologists studying the problem of chemical carcinogenesis. On the other hand, there is a strong need for elaborating a standardized test that would explicitly define the carcinogenic properties of a specific compound. The use of a variety of methods for the determination of carcinogenic potency of chemical compounds combined with simultaneous differentiation of structure of examined compounds, sometimes leads to contradictory conclusions. Correlation between mutagenic and carcinogenic activity of some compounds is also sometimes questionable. Modern molecular biology should allow for elaborating a method which would give a satisfactory and reliable answer to the fundamental question: is a specific compound carcinogenous for a human organism? And if so - to what extent

Cyclopenta[c]phenanthrene induction of CYP1A in brain of rainbow trout (Oncorhynchus mykiss)

We assessed the effects of cyclopenta[c]phenanthrene (CP[c]Ph) and benzo[a]pyrene (B[a]P; positive control) on CYP1A gene expression in brain of juvenile rainbow trout (Oncorhynchus mykiss) using the quantitative reverse transcription polymerase chain reaction (Q-RT-PCR). A group of hatchery raised rainbow trout, with an average body mass of 49.4 g and total length of 15.5 cm were given an intraperitoneal injection (10 mg*kg-1) of either CP[c]Ph or B[a]P in corn oil (2 mg*mi-1 corn oil) or corn oil alone (control). After 24 and 48 h, trout brains were collected for mRNA isolation and analysis. After 24 hours of the exposure, only B[a]P-treated rainbow trout had 10-fold higher number of CYP1A transcripts (mean = 3.63*106 transcripts*µg-1 total RNA) than control fish (3.24*105 transcripts*µg-1 total RNA; Tukey test, P<0.05). After 48 hrs, significantly higher levels of CYP1A expression (Tukey test, P<0.001) were found in either CP[c]Ph- or B[a]P- induced group (1.45*106 and 6.92*106 transcriptsźµg-1 total RNA, respectively) over a control group (mean=1.41*105 transcripts*µg-1 total RNA). The finding that CYP1A in brain tissue was inducible by CP[c]Ph, a polycyclic aromatic hydrocarbon (PAH) of different than B[a]P planar characteristics, may further validate the use of rainbow trout brain CYP1A mRNA levels as a biomarker of PAH exposure

Preliminary evaluation of mutagenic activity of two amino derivatives of cyclopenta[c]phenanthrene

The mutagenicity of two, newly synthesized amino-derivatives of cyclopenta[c]phenanthrene (CP[c]Ph) was investigated in the Ames test using TA98 and TA100 histidine dependent Salmonella typhimurium strains. Neither of the examined compounds showed mutagenic activity without metabolic activation. The incorporation of an activation system caused high mutagenicity of CP[c]Ph derivatives in both S. typhimurium strains. The results are compared with the previous data on the mutagenic activity of unsubstituted CP[c]Ph and possible mechanisms of activation are discussed

2,4-diphenylthiophene induces mainly base pair mutation in Salmonella Typhimurium

Heterocyclic aromatic compounds containing sulfur (S-HET), have been detected in air, soil, marine environment and freshwater sediment. Toxicity and mutagenicity data of this class of substances are scarce. The present study focuses on implications of two aryl thiophenes and their mutagenic properties in Salmonella/microsome test. In our experiment only 2,4-diphenylthiophene showed little mutagenic effect in both variants of activaction (+/-S9) in strain TA100. Thiophene ring joined to K-region of phenanthrene did not change the biological activity of 3,6-dimetoxyphenanthro [9,10-c]thiophene and this compound did not show mutagenic potency

Mutagenicity induced in Salmonella strains TA98 and TA100 by diphenylthiophenes

Mutagenic properties of four different diphenylthiophenes: 3,4-diphenylthiophene, 3,4-di(4'-methylphenyl)thiophene, 3,4-di(4'-methoxyphenyl)thiophene and 3,4-di(4'-pentoxyphenyl)thiophene were investigated applying the Salmonella test. The research was done on two strains of Salmonella Typhimurium: TA98 and TA100, tested in two variants: with (+S9) and without (-S9) enzymatic activation. Only one compound 3,4-di(4'-methylphenyl)thiophene showed mutagenic activity when studied with metabolic activation (+S9) and its mutagenic rate (MR) score was 3.41 for the dose of 10.00µg*plate-1. Other studied compounds did not show any mutagenic activity (+/-S9) and their MR score did not exceed the threshold value of 2.0

Preliminary study on adverse effects of phenanthrene and its methyl and phenyl derivatives in larval zebrafish, Danio rerio

Toxic effects of polycyclic aromatic hydrocarbons (PAHs) have been extensively studied in fish, although knowledge concerning biological activities of phenanthrene and its derivatives remains still incomplete. The aim of this work was to evaluate lethal and sublethal effects of benzo(a)pyrene, phenanthrene and phenanthrene derivatives (1-methylphenanthrene, 4-methylphenanthrene, 1-phenylphenanthrene and 4-phenylphenanthrene) on zebrafish (Danio rerio) larvae. We conducted acute toxicity test, using 96h static renewal exposure to a series of the PAH concentrations (0.05, 0.50, 5.00, 50.00µmol*l-1), to determine the No Effect Concentration (NEC) value for each compound examined. The mean NEC estimates obtained in the study were 5.16۪.45µmol*l-1 (B[a]P), 4.88۪.13µmol*l-1 (Ph), 40.24䔰.93µmol*l-1 (1P-Ph), 47.92ۭ.61µmol*l-1 (1M-Ph), 24.31۱.33µmol*l-1 (4P-Ph) and 3.11۫.01µmol*l-1 (4M-Ph) and suggested the following order of PAH toxicities on Danio rerio larvae: 4M-Ph>Ph˜B[a]P>4PPhP-Ph>1M-Ph. To gain insight into possible molecular mechanisms of apparent toxicity of phenanthrene derivatives on zebrafish larvae, we examined mRNA expression of cyp1a, cyp1b1, and vtg genes in the larvae exposed for 48h to a PAH concentration of 0.50µmol*l-1. Whereas the larvae exposed to each tested PAH displayed many developmental abnormalities (i.e. pericardial and yolk sac edema, dorsal curvature, or tail malformations), no significant upregulation of cyp1a and cyp1b1 mRNA was observed, except for zebrafish exposed to B[a]P. However, significant reduction of vtg mRNA was observed in the larvae exposed to phenanthrene and its 4P- derivative. The results may contribute to the development of a new knowledge about effects of structurally diverse phenanthrene derivatives on vertebrate organisms