Multiphoton spectral analysis of benzo[a]pyrene uptake and metabolism in a rat liver cell line

Dynamic analysis of the uptake and metabolism of polycyclic aromatic hydrocarbons (PAHs) and their metabolites within live cells in real time has the potential to provide novel insights into genotoxic and non-genotoxic mechanisms of cellular injury caused by PAHs. The present work, combining the use of metabolite spectra generated from metabolite standards using multiphoton spectral analysis and an “advanced unmixing process”, identifies and quantifies the uptake, partitioning, and metabolite formation of one of the most important PAHs (benzo[a]pyrene, BaP) in viable cultured rat liver cells over a period of 24 h. The application of the advanced unmixing process resulted in the simultaneous identification of 8 metabolites in live cells at any single time. The accuracy of this unmixing process was verified using specific microsomal epoxide hydrolase inhibitors, glucuronidation and sulfation inhibitors as well as several mixtures of metabolite standards. Our findings prove that the two-photon microscopy imaging surpasses the conventional fluorescence imaging techniques and the unmixing process is a mathematical technique that seems applicable to the analysis of BaP metabolites in living cells especially for analysis of changes of the ultimate carcinogen benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide. Therefore, the combination of the two-photon acquisition with the unmixing process should provide important insights into the cellular and molecular mechanisms by which BaP and other PAHs alter cellular homeostasis.     

Genotoxic effects of benzo[a]pyrene and dibenzo[a,l]pyrene in a human lung cell line

Several studies have shown that polycyclic aromatic hydrocarbons (PAHs) produce genotoxic effects in assays performed in vivo and in vitro. This study was undertaken to investigate the ability of benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) to induce DNA damage in a human lung fibroblast cell line (MRC-5), using sister-chromatid exchanges test (SCEs), the comet assay, and evaluating point mutations in codon 12 of the K-ras protooncogene by polymerase chain reaction-single-strand conformation polymorphisms (PCR-SSCPs) and restriction fragment length polymorphisms (RFLP)-enriched PCR methods. Sister-chromatid exchanges frequencies were significantly increased in cells exposed to benzo[a]pyrene and dibenzo[a,l]pyrene in relation to controls (p < .001). Using the standard alkaline comet assay, significant differences between groups were found for the variable comet moment (CM) when cells were exposed to BP (p < .001) and DBP (p < .001). Nevertheless, PCR-SSCP and RFLP-enriched PCR methods did not show any association between treatments with BP and DBP and K-ras point mutations. The data presented in this study indicated that BP and DBP induced both DNA strand breaks and sister-chromatid exchanges but not significant point mutations at codon 12 of K-ras gene in the MRC-5 cell line.     

Evaluation of murine splenic cell type metabolism of benzo[a]pyrene and functionality in vitro following repeated in vivo exposure to benzo[a]pyrene.

Recent studies have demonstrated that macrophages are the cell types capable of metabolizing benzo[a]pyrene (B(a)P) within the spleens of untreated mice. Since repeated exposure to B(a)P results in immunosuppression and B(a)P is known to induce cytochrome P450 levels, the first objective of this study was to investigate whether exposure of mice to B(a)P could increase the amounts of immunosuppressive B(a)P metabolites generated and/or alter the pattern of B(a)P metabolites formed by several different splenic cell types. Mice were dosed with a daily sc dose of 200 mg/kg B(a)P or vehicle for 4 days. Separation of splenocytes based on density by centrifugation through discontinuous Percoll gradients along with immunomagnetic negative selection or antibody-mediated complement lysis was used to obtain different splenic cell populations. Cells were incubated with [3H]B(a)P for 24 hr. High-pressure liquid chromatography was used to separate and quantitate B(a)P metabolites. Results indicate that splenic macrophages of B(a)P-treated mice produced significantly greater amounts of some metabolites compared to those of vehicle-treated mice. The three major metabolites produced were an unidentified peak of polar metabolites containing polyhydroxylated metabolites, B(a)P-9,10- and B(a)P-7,8-dihydrodiols. Other splenic cell types examined did not produce metabolite amounts significantly above (T-cells, PMNs, or the capsule) or just above (B-cells) background. The second objective was to investigate the splenic cell type(s) targeted by B(a)P resulting in suppression of humoral immunity. Separation-reconstitution studies along with in vitro sensitization techniques with several different antigens (sheep red blood cells (SRBC), dinitrophenyl-Ficoll (DNP-Ficoll), lipopolysaccharide (LPS)) were used to identify splenic target cells following exposure of mice to B(a)P (200 mg/kg/day, sc for 4 days). Findings indicate that in vitro plaque-forming cell (PFC) suppression was due to alterations in the adherent (macrophage) cell population. Exposure also suppressed the PFC response to the T-dependent antigen SRBC and the T-independent antigen DNP-Ficoll, but did not suppress the PFC response to the polyclonal antigen, LPS. These data suggest that B(a)P is targeting macrophages.     

Using ovarian and brain cell culture from the Mullet,Liza klunzingeri,to assess the inhibitory effects of benzo[a]pyrene on aromatase activity

We assessed the toxic effects of benzo[a]pyrene (BaP) on cell viability, aromatase (Aro) activity and steroid production using ovarian and brain cell cultures obtained from Mullet, Liza klunzingeri. The brain and ovary were minced and digested, and the cells were suspended in Leibovitz's L-15 medium supplemented with 15% and 20% fetal bovine serum. The cell suspensions were seeded on 25-cm² cell-culture flasks at 1 × 10⁶ cells/mL and incubated at 25 °C for 2 weeks. A BaP concentration of 10⁻⁵ mol/L was accepted as the half-maximal inhibitory concentration. Ovarian and brain cells were exposed to different concentrations of BaP [0 (control), 10⁻⁶, 2 × 10⁻⁶, 3 × 10⁻⁶ mol/L] and incubated at 30 °C. At different sampling times (0, 12, 24 and 48 h) 40 ng/10⁵ cells of 1,4,6-androstatriene-3,17-dione (ATD) was added to each well. Aro activity, 17β-estradiol (E2) and ATD production were determined. The sensitivity of the cultivated ovarian and brain cells to BaP increased dose dependently. BaP was a potent inhibitor of Aro activity at 2 × 10⁻⁶ and 3 × 10⁻⁶ mol/L, both in the cultivated brain and ovarian cells at different sampling times, with 10⁻⁶ mol/L BaP found to be the least potent Aro inhibitor. E2 production decreased from cultivated ovarian and brain cells treated by different concentrations of BaP. In conclusion, BaP is able to change the activity of Aro and disrupt the biosynthesis of estrogens, and thus affects reproduction in fish.     

Single and concerted effects of benzo[a]pyrene and flavonoids on the AhR and Nrf2-pathway in the human colon carcinoma cell line Caco-2

As phytochemicals have the potential to counteract adverse effects of carcinogens we investigated the influence of the flavonoids quercetin and kaempferol on benzo[a]pyrene (BaP) mediated effects on human colon cancer cells, Caco-2. We focused on concerted effects on the expression of AhR and Nrf2 pathway components. In contrast to kaempferol, BaP and quercetin efficiently induced CYP1A1, CYP1A2 and CYP1B1-mRNA in Caco-2 cells. BaP not only acted via AhR activation but sustainably also by increasing AhR and by down-regulating AhRR mRNA. The flavonoids did not affect AhR expression but counteracted the BaP mediated AhRR repression. Only quercetin was found to induce AhRR mRNA. ARNT mRNA appeared to be slightly but significantly down-regulated by BaP as well as by flavonoids while expression of AIP was not or only slightly modulated. The Nrf2 pathway was activated by BaP and by the flavonoids shown by induction of Nrf2 and several of its target genes such as NQO1, GSTP1, GSTA1 and GCLC. Induction effects of 10 μm BaP on Nrf2, GSTP1 and NQO1 were abolished by the flavonoids. In summary, we show that quercetin supports AhR mediated effects. Both flavonoids, however, may counteract the effects of BaP on expression of AhR, AhRR, Nrf2, GSTP1 and NQO1. In conclusion, quercetin appears to have two faces, a flavonoid-like one and a PAH-like one which supports Ahr-mediated effects while kaempferol acts “just like a flavonoid”. Thus, flavonoids have to be treated individually with respect to their anti-adverse activity.     

Studies on the ability of rat lung and liver microsomes to facilitate transfer and metabolism of benzo[a]pyrene from diesel particles

Little is known about the bioavailability of inhaled organic compounds that are associated with particles. It is known that certain particle-associated organic compounds, such as polycyclic aromatic hydrocarbons (PAH) adsorbed on diesel soot particles, are retained in the lung longer than PAH inhaled in pure form. If such particle-associated compounds are available for tissue interaction, their prolonged retention may result in an increased potential for a toxic effect. To determine the factors affecting the bioavailability of particle-associated PAH, we have studied the ability of microsomes to facilitate transfer of benzo[a]pyrene (B[a]P) adsorbed on the surface of diesel exhaust soot particles to the microsomes and the ability of the microsomes to metabolize the transferred B[a]P. Our results indicate that rat lung and liver microsomes were able to facilitate the transfer of small amounts of B[a]P from diesel particles (less than 3%), but only a fraction of the amount transferred (1-2%) was metabolized. Under the same incubation conditions without soot, free B[a]P was extensively metabolized by microsomes, principally to B[a]P-9,10-diol. Lung microsomes were about twice as effective as liver microsomes for the transfer of the B[a]P. The ability to transfer B[a]P to the microsomes was independent of metabolism or the presence of protein, but was related to the lipid content of the microsomal fraction. There was no metabolism of the B[a]P coated on diesel particles as analyzed by high-performance liquid chromatography. These findings suggest that microsomes are able to enhance the slow transfer of only a small amount of B[a]P from diesel particles in a form that can be metabolized. However, over a long period of time, this slow release might be significant.     

Comparison of passive-dosed and solvent spiked exposures of pro-carcinogen,benzo[a]pyrene,to human lymphoblastoid cell line,MCL-5

Genotoxicity testing methods in vitro provide a means to predict the DNA damaging effects of chemicals on human cells. This is hindered in the case of hydrophobic test compounds, however, which will partition to in vitro components such as plastic-ware and medium proteins, in preference to the aqueous phase of the exposure medium. This affects the freely available test chemical concentration, and as this freely dissolved aqueous concentration is that bioavailable to cells, it is important to define and maintain this exposure. Passive dosing promises to have an advantage over traditional ‘solvent spiking’ exposure methods and involves the establishment and maintenance of known chemical concentrations in the in vitro medium, and therefore aqueous phase. Passive dosing was applied in a novel format to expose the MCL-5 human lymphoblastoid cell line to the pro-carcinogen, benzo[a]pyrene (B[a]P) and was compared to solvent (dimethyl sulphoxide) spiked B[a]P exposures over 48 h. Passive dosing induced greater changes, at lower concentrations, to micronucleus frequency, p21 mRNA expression, cell cycle abnormalities, and cell and nuclear morphology. This was attributed to a maintained, definable, free chemical concentration using passive dosing and the presence or absence of solvent, and highlights the influence of exposure choice on genotoxic outcomes.     

Radical cations in the horseradish peroxidase and prostaglandin H synthase mediated metabolism and binding of benzo[a]pyrene to deoxyribonucleic acid

Metabolism and DNA binding studies are used to investigate mechanisms of activation for carcinogens. In this paper we describe metabolism of benzo[a]pyrene (BP) and 6-fluorobenzo[a]pyrene (6-FBP) by two peroxidases, horseradish peroxidase (HRP) and prostaglandin H synthase (PHS), which are known to catalyze one-electron oxidation. In addition, binding of BP and BP quinones to DNA was compared in the two enzyme systems. The only metabolites formed from BP or 6-FBP by either enzyme were the quinones, BP 1,6-, 3,6- and 6,12-dione. HRP metabolized BP and 6-FBP to the same extent and produced the same proportion of each dione from both compounds, approximately 40% each of BP 1,6- and 3,6-dione and 20% BP 6,12-dione. PHS formed twice as much quinones from BP as from 6-FBP and produced relatively more BP 3,6-dione from 6-FBP (46%) compared to BP (30%) and relatively less BP 6,12-dione from 6-FBP (16%) compared to BP (33%). Removal of the fluoro substituent in the metabolism of 6-FBP is consistent only with an initial one-electron oxidation of the substrate. Since BP quinones were the only products formed in HRP- and PHS-catalyzed activation of BP, their possible binding to DNA was compared to that of BP. No significant binding of BP quinones to DNA occurred with either HRP or PHS. These results, coupled with those from other chemical and biochemical experiments, demonstrate that HRP- and PHS-catalyzed one-electron oxidation of BP to its radical cation is the mechanism of formation of quinones and binding of BP to DNA.     

Ah receptor mediating induction of cytochrome P450IA1 in a novel continuous human liver cell line (Mz-Hep-1). Detection by binding with [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin and relationship to the activity of aryl hydrocarbon hydroxylase.

The Ah receptor regulates induction of cytochrome P450IA1 and mediates certain toxicities of polyhalogenated aromatics such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). It has been characterized previously in continuous cell lines, notably the mouse hepatoma line Hepa 1, the human squamous cell carcinoma line A431, and the human liver cell line Hep G2. The present work extends our knowledge of the Ah receptor in continuous human liver cell lines. Ah receptor can be detected in Mz-Hep-1, a hepatitis B virus-negative cell line derived from a Thorotrast-induced hepatocellular carcinoma. The mean concentration of Ah receptor in Mz-Hep-1 cells was 341 +/- 22 fmol/mg cytosol protein (mean +/- SEM, nine separate determinations). This is equivalent to approximately 30,000 sites per cell. The concentration of Ah receptor in Mz-Hep-1 cells is similar to that in Hepa 1 cells and approximately three times higher than that in Hep G2 cells. The Mz-Hep-1 Ah receptor sedimented in continuous sucrose gradients at approximately 9 S. Specificity of binding by [3H]TCDD was demonstrated by competitive binding of non-radiolabeled 2,3,7,8-tetrachlorodibenzofuran, 3-methylcholanthrene (MC), and dibenz[a,h]anthracene in 50-fold molar excess. Phenobarbital, which is not a substrate for P450IA1, did not compete with [3H]TCDD for binding to Mz-Hep-1 Ah receptor. Dexamethasone and estradiol also did not compete with [3H]TCDD for binding, suggesting non-identity of Ah receptor with glucocorticoid or estrogen receptor. In separate experiments, glucocorticoid receptor was identified in Mz-Hep-1 cells. By Scatchard plot analysis, the apparent equilibrium dissociation constant (Kd) for binding of [3H]TCDD to Mz-Hep-1 Ah receptor was estimated to be 4.4 nM, compared to 0.8 nM in Hepa 1 cells. By Woolf plot analysis the Kd was 5.4 nM, compared to 1.2 nM in Hepa 1 cells. The [3H]TCDD.Ah receptor complex extracted from nuclei of Mz-Hep-1 cells incubated with [3H]TCDD in culture at 37 degrees sedimented at approximately 6 S under conditions of high ionic strength. Aryl hydrocarbon hydroxylase (AHH) activity was detectable in Mz-Hep-1 cells after pretreatment with inducing chemicals. Mz-Hep-1 cells have the highest concentrations of Ah receptor in any continuous human liver cell line thus far investigated. The Mz-Hep-1 Ah receptor is similar physicochemically to that described in murine systems. AHH activity is inducible in Mz-Hep-1 cells.