Benzo[b]fluoranthene (B[b]F) affects apoptosis,oxidative stress,mitochondrial membrane potential and expressions of blood-brain barrier markers in microvascular endothelial cells

Exposure to polycyclic aromatic hydrocarbons (PAHs) contributes to the damage of blood-brain barrier. While a number of studies were focused on benzo[a]pyrene, direct effects and mechanisms of benzo[b]fluoranthene (B[b]F), another main component of PAHs, on blood-brain barrier (BBB) are not documented. Here, we investigated if B[b]F at concentrations of environmental relevance could affect apoptosis, oxidative stress, mitochondrial membrane potential (MMP) and BBB marker expression in mouse brain microvascular endothelial (bEnd.3) cells, an in vitro model typically used to study BBB toxicology. Cells were treated with varying concentrations of B[b]F (0, 10, 20 and 40 μM) for 48 h. Cell proliferation, cell cycle, apoptosis, oxidative stress, MMP and BBB marker expressions were evaluated by label-free real-time cell analysis, flow cytometry, immunofluorescence and Western-blot. The proliferation of bEnd.3 cells was inhibited by B[b]F in a concentration dependent manner. B[b]F treatment significantly affected cell cycle, induced apoptosis, increased levels of reactive oxygen species (ROS) and disputed MMP. Expressions of BBB marker Occludin and Claudin-5 were decreased in the presence of 40 μM B[b]F. In conclusion, B[b]F might damage BBB by affecting proliferation, apoptosis, ROS level and Occludin and Claudin-5 expressions in microvascular endothelial cells.     

Effect of carcinogenic polycyclic aromatic hydrocarbons on mouse embryonic cells in culture: Induction of spindle-shaped cells

In cultured mouse embroyonic cells (MECs) treated with benzo[a]pyrene(B[a]P), there appeared unusual type of fibroblasts, spindle-shaped cells (SP cells), which were characterized by their narrow bipolar shape, long cellular processes and optically distinct cell borders. Appearance of SP cells was massive and irreversible. The amount of SP cells increased with increased with increasing concentrations of B[a]P, while early cytotoxicity did not. In various polycyclic aromatic hydrocarbons (PAHs) tested, only potent carcinogens {7,12-dimethylabenz[a]anthracene (DMBA), 3-methylcholanthrene (MCA), B[a]P and dibenz[a, e]pyrene (DB[a, e]P)} induced SP cells. Among them, PAH having higher Iball's index induced SP cells at lower concentration and at an earlier time. Weak or non-carcinogenic PAHs including 3-hydroxybenzo-[a]-pyrene(3-H-B[a]P) did not induce SP cells. α-Napthoflavon (αNF) suppressed the induction of SP cell by carcinogenic PAH. SP cells did not appear spontaneously under various abnormal culture conditions. These results indicate that carcinogenic PAHs induce the appearance of a specific type of fibroblast, SP cells in MEC cultures in accordance with their carcinogenicity.     

Toxicity and EROD-inducing potency of 24 polycyclic aromatic hydrocarbons (PAHs) in chick embryos

The toxicities (embryolethality) of 24 polycyclic aromatic hydrocarbons (PAHs) were determined in chick embryos using a 72-h test. The substances, dissolved in peanut oil, were injected into the air sacs of eggs preincubated for 7 days. LD₅₀ values were determined for the four most toxic of the 24 compounds. Benzo [k] fluoranthene proved to be the most potent, with an LD₅₀ of 14 g (56 nmol)/kg egg. Dibenz[a,h]anthracene, benz[a]anthracene and benzo[b]naphtho[2,3-d]thiophene were a few times less toxic [LD₅₀=39 g (140 nmol)/kg, 79 g (349 nmol)/kg and 82 g (350 nmol)/kg, respectively]. The LD₅₀ of benzo [k] fluoranthene was only about 5 times higher than that previously found for the most potent coplanar polychlorinated biphenyl (PCB), 3,3,4,4,5-pentachlorobiphenyl [LD₅₀=3.1 g (9.4 nmol)/kg], in the same kind of test. The toxicities of 18 of the PAHs in this study have also been evaluated previously using a 2-week test in chick embryos. Dibenz[a,h]anthracene, which had not been studied earlier in the 2-week test, proved to be almost as toxic as previously found for benzo [k] fluoranthene in that test. Several of the PAHs studied induced EROD activity in chick embryos, and, in general, the most toxic PAHs were also the most potent inducers of EROD. The highest enzyme activities were found after treatment with indeno[1,2,3-c,d]pyrene (12 times the control value) and dibenz[a,h]anthracene (8 times the control value). However, due to the high toxicity of dibenz[a,h]anthracene, the dose used was 7 times lower than that of indeno [1,2,3-c,d]pyrene. Following injection of PAHs on day 7, the EROD activities on day 10 were considerably lower than those obtained after a corresponding treatment with coplanar PCBs in an earlier study. Of the PAHs studied, some exhibited very high embryotoxicity. The most toxic PAHs induced EROD activity, suggesting that their toxicity was at least partly mediated via binding to the Ah receptor.     

Effects of two oils and 16 pure polycyclic aromatic hydrocarbons on plasmatic immune parameters in the European sea bass,Dicentrarchus labrax (Linné)

The in vitro effects of polycyclic aromatic hydrocarbons (PAHs) on two plasmatic immune parameters, lysozyme concentration and haemolytic alternative complement activity, of the European sea bass, Dicentrarchus labrax, were tested using field (10^?7 and 10^?9 mg mL^?1) and high concentrations (10^?3 and 10^?5 mg mL^?1) observed during oil spills. Peripheral blood from 105 fish was collected, centrifuged at 1200g, for 10 min, at 4 °C and three plasma pools, each of 35 fish, were constituted. Two oils (heavy fuel oil and light cycle oil) and 16 pure PAHs, selected on the basis of the American Environmental Protection Agency list (US EPA), were tested in vitro on the two humoral immune parameters. Only three pure PAHs (anthracene, chrysene and dibenz[a,h]anthracene) modulated lysozyme concentration. Acenaphthene, acenaphthylene, anthracene, benzo[a]anthracene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, pyrene and light cycle oil modified the haemolytic alternative complement activity after 4 h of incubation. This study investigates the direct effects of several PAHs on fish humoral immune functions and describes the haemolytic complement activity of fish as suitable biomarkers of oil pollution.     

Effects of naphthalene on benzo[a]pyrene hydroxylase and cytochrome P-450 in Fundulus heteroclitus

Hepatic benzo[a]pyrene (B[a]P) hydroxylase, cytochrome P-450 and cytochrome b₅ were investigated in the mummichog, Fundulus heteroclitus, following acute exposure to naphthalene dissolved in the water. Control experiments revealed that males had significantly higher levels of BaP hydroxylase and cytochrome P-450 compared to females. Significant variation of B[a]P hydroxylase activity was also observed in control fish during a 6-mth period which may reflect seasonal or reproductive influences.Naphthalene at a concentration of 4 mg/l caused a significant reduction of B[a]P hydroxylase activity in males and females. The naphthalene exposure effected significant decreases of cytochrome P-450 in males, but not in females. Addition of naphthalene directly to reaction mixtures containing liver preparation from control fish caused reduced B[a]P hydroxylase activity in vitro. The concentrations of naphthalene necessary to produce in vitro reductions in B[a]P hydroxylase appeared to be much higher than would be realized by in vivo exposures. These data indicate that metabolite formation or other mechanisms such as generalized physiological stress as a result of in vivo exposures may be important factors in reducing B[a]P hydroxylase activity. The nature of the reduction of B[a]P hydroxylase was not elucidated. Cytochrome b₅ concentrations did not change significantly.     

Human exposure to polycyclic aromatic hydrocarbons (PAHs) using data from a duplicate diet study in Catalonia,Spain

In this study, the dietary intake of 16 polycyclic aromatic hydrocarbons (PAHs) by the population of Tarragona County (Catalonia, Spain) was assessed using the duplicate diet approach. Duplicate diet samples, prepared as per consumption, were collected during September 2010 in various restaurants offering a variety of daily menus (breakfast, lunch, and dinner). For analysis of PAHs, a total of 90 composite samples were prepared. Analytical procedure of PAHs was performed by means of gas chromatography/mass spectrometry. Intake calculations were made for the standard male adult population. The highest intakes corresponded to acenaphthylene (12.7 μg/day), acenaphthene (12.4 μg/day), and fluorene (11.9 μg/day), while the lowest intake corresponded to dibenz[a,h]anthracene (0.12 μg/day), being also comparatively low those of pyrene, benzo[b]fluoranthene + benzo[j]fluoranthene, benzo[a]pyrene and benzo[ghi]perylene (0.13 μg/day in all cases). The results were compared with data from previous total diet studies (TDS) recently performed in the same geographical area. In the present study, the estimated mean dietary intake for a standard male adult living in Catalonia was 59.2 μg/day, a value notably higher than that found in our recent TDS (6.72 μg/day). However, it is essential to remark that important methodological differences exist between both surveys, reflecting that calculation methods should be similar when the purpose is to compare results from different surveys. In general terms, we conclude that for PAHs, duplicate diet studies may be a good alternative to total diet studies, especially when there are important economical limitations to perform a suitable TDS. The costs associated to the former may be notably lower, as they do not require such an extensive number of samples for chemical analysis. Moreover, a duplicate diet approach may even be more realistic, as cooked foodstuffs are used for dietary exposure assessment.     

Effects of benzo[a]pyrene exposure on oxidative stress and apoptosis of gill cells of Chlamys farreri in vitro

As a common pollutant in marine environment, benzo[a]pyrene (B[a]P) has high toxicity to economic shellfish. In order to explore the mechanism of oxidative stress and apoptosis, the effects of 0, 2, 4, 8 μg/mL B[a]P on gill cells of C. farreri at 12 and 24 h were studied. The results showed that B[a]P decreased the activity of gill cells, increased the content of reactive oxygen species (ROS) and the expression of antioxidant defense genes. Besides, B[a]P could induce oxidative damage to nucleus and mitochondria. The gene expression and enzyme activity of apoptosis pathway related factors were changed. In conclusion, these results showed that B[a]P could cause oxidative stress and oxidative damage in gill cells of C. farreri, and mediate gill cell apoptosis through mitochondrial pathway and death receptor pathway. This article provides a theoretical basis for clarifying the molecular mechanism of PAHs-included oxidative stress and apoptosis in bivalves.     

Quantitative microspectrofluorimetry study of the “blocking effect” of 6-aminochrysene on benzo[a]pyrene metabolism using single living cells

By microspectrofluorimetry on single living cells (murine fibroblasts 3T3), we have obtained monoexponential decreases of fluorescence intensity for benzo[a]pyrene (B[a]P) and 6-aminochrysene (6a-chrysene) metabolism. These kinetics are characteristics of B[a]P and 6a-chrysene metabolism and histograms can be drawn from the rate constants. We have studied the influence of 6a-chrysene on B[a]P metabolism. Using different methods of incubation, it has been observed that the presence of 6a-chrysene leads to modifications of the histogram profiles during B[a]P metabolism. Polycyclic aromatic hydrocarbons (PAH) are used to induce B[a]P metabolism. Whatever the experimental conditions we never detected such a phenomenon with 6a-chrysene. On the contrary we have observed an inhibition of B[a]P metabolism by 6a-chrysese, which can reach 80% of the aryl hydrocarbon hydroxylase (AHH) activity when 6a-chrysene remains constant in the cells. Compared with the results previously observed “in vitro” (which presented 50% mean inhibition) we show that inhibiyion acts in an all-or-nothing mechanism at the cellular level.     

Inflammatory mediators accelerate metabolism of benzo[a]pyrene in rat alveolar type II cells: The role of enhanced cytochrome P450 1B1 expression

Long-term deregulated inflammation represents one of the key factors contributing to lung cancer etiology. Previously, we have observed that tumor necrosis factor-α (TNF-α), a major pro-inflammatory cytokine, enhances genotoxicity of benzo[a]pyrene (B[a]P), a highly carcinogenic polycyclic aromatic hydrocarbon, in rat lung epithelial RLE-6TN cells, a model of alveolar type II cells. Therefore, we analyzed B[a]P metabolism in RLE-6TN cells under inflammatory conditions, simulated using either recombinant TNF-α, or a mixture of inflammatory mediators derived from activated alveolar macrophage cell line. Inflammatory conditions significantly accelerated BaP metabolism, as evidenced by decreased levels of both parent B[a]P and its metabolites. TNF-α altered production of the metabolites associated with dihydrodiol-epoxide and radical cation pathways of B[a]P metabolism, especially B[a]P-dihydrodiols, and B[a]P-diones. We then evaluated the role of cytochrome P450 1B1 (CYP1B1), which is strongly up-regulated in cells treated with B[a]P under inflammatory conditions, in the observed effects. The siRNA-mediated CYP1B1 knock-down increased levels of B[a]P and reduced formation of stable DNA adducts, thus confirming the essential role of CYP1B1 in B[a]P metabolism under inflammatory conditions. TNF-α also reduced expression of aldo-keto reductase 1C14, which may compete with CYP1B1 for B[a]P-7,8-dihydrodiol and divert it from the formation of ultimate B[a]P dihydrodiol epoxide. Together, the present data suggests that the CYP1B1-catalyzed metabolism of polycyclic aromatic hydrocarbons might contribute to their enhanced bioactivation and genotoxic effects under inflammatory conditions.     

Modulation of CYP1A1 activity by a Ginkgo biloba extract in the human intestinal Caco-2 cells

Ginkgo biloba is a widely consumed dietary supplement. Some dietary active compounds modulate the activity of biotransformation enzymes inside the enterocytes and more interestingly of cytochrome P-450 1A1 (CYP1A1). This enzyme is of a particular interest because of its implication in the metabolism of some exogenous pro-carcinogens or endogenous molecules. In the present work, we have used Caco-2 cells to study the effect of a standard reference material of a Ginkgo biloba extract (GBE) (10-400 μg/ml), as well as of its major individual active compounds (kaempferol, quercetin, isorhamnetin, ginkgolides and bilobalide), alone or in mixtures, at realistic intestinal concentrations, on the induction of CYP1A1 activity, in the presence or absence of benzo[a]pyrene (B[a]P) (0.1 μg/ml), a well-known CYP1A1 inducer. 3-O-rutinosides of kaempferol, quercetin and isorhamnetin were also tested. We have demonstrated a strong induction (p < 0.005) of CYP1A1 activity and a slight, but significant (p < 0.005), decrease of this activity in the presence of B[a]P by the GBE at the realistic exposure level of 100 μg/ml. The inductive effect was explained, in part, by quercetin and kaempferol after 24 h exposure while unknown compounds seem to be responsible for the strong CYP1A1 induction observed after 6 h exposure. The inhibitory potency of flavonols on CYP1A1 activity in presence of B[a]P was much stronger for the aglycones than for the 3-O-rutinosides, explaining the slight effect observed with the GBE, mainly composed of glycosylated flavonoids. These results indicate that GBEs may disturb intestinal CYP1A1 activity and, in turn, affect the metabolism of other compounds. The present paper thus highlights the necessity to take these side effects into account when administrating Ginkgo biloba herbal supplements.     

Metabolism and excretion of benzo[a]pyrene in the rabbit

1. Following i.v. administration of [¹⁴C]benzo[a]pyrene (3 μmol/kg) to rabbits, 30% of the ¹⁴C dose appeared in bile and 12% in urine, within six hours.

2. Biliary and urinary metabolites were mainly conjugated; <12% of the ¹⁴C was extractable with ethyl acetate, but after treatment with β-glucuronidase or aryl sulphatase 30–40% became extractable.

3. H.p.l.c. analysis of the extracts indicated that the major non-polar metabolite was benzo[a]pyrene, 9,10-diol (18% of ¹⁴C in bile and 24% of ¹⁴C in urine, mainly conjugated with glucuronic acid). Smaller amounts of the 4,5-diol, the 3,6-quinone, and the 9-hydroxy- and 3-hydroxybenzo[a]pyrene were also found in bile (total <10%), together with 9-hydroxybenzo[a]pyrene and two unknown metabolites (X and Y) in urine (total <4%).

4. The proximate carcinogen, the 7,8-diol, was not detected in any extract.

5. After intraduodenal administration of biliary metabolites of [¹⁴C]benzo[a]pyrene (approx. 0·3 μmol), ¹⁴C was excreted in the bile (21% dose) and urine (14%) within 23?h, indicating that metabolites can undergo enterohepatic circulation in the rabbit.     

Kinetic modelling of in vitro cell-based assays to characterize non-specific bindings and ADME processes in a static and a perfused fluidic system

Recently, physiologically based perfusion in vitro systems have been developed to provide cell culture environment close to in vivo cell environment (e.g., fluidic conditions, organ interactions). In this work, we model and compare the fate of a chemical, benzo[a]pyrene (B[a]P), in a perfusion and a standard (static well-plate) system. These in vitro systems are composed of Caco-2 and HepG2 cells so as to mimic absorption across the small intestine and intestinal and hepatic metabolism. Compartmental models were developed and calibrated with B[a]P kinetics data in the culture medium to estimate the apparent permeability of Caco-2 cells, the in vitro biotransformation of B[a]P, as well as the different routes of loss by non-specific adsorption. Our results show that non-specific binding is the main process responsible for the depletion of B[a]P in the culture media: at steady state, only 40% and 24% of the total concentration of B[a]P are bioavailable in the static and perfused systems, respectively. We also showed that Caco-2 permeability in the perfused culture system is closer to in vivo conditions than the one obtained in the static system and that higher cellular metabolic activities are observed in static conditions. Perfused in vitro systems combined with kinetic modelling are promising tools for studying in vitro the different processes involved in the toxicokinetics of xenobiotics.     

Importance of CYP1A1 and CYP1B1 in bioactivation of benzo[a]pyrene in human lung cell lines

Polycyclic aromatic hydrocarbons are ubiquitous environmental pollutants classified as carcinogens in humans and rodents. The cytochromes P4501A1 and 1B1 have both shown capacity to carry out bioactivation of the prototype PAH, benzo[a]pyrene (B[a]P) to its ultimate carcinogenic B[a]P-diol-epoxide-I-1 form. The part played by each enzyme in human lung cells, however, has not been clarified. To get further insight into their individual role in the metabolic activation of B[a]P, RNA-interference was used to down-regulate CYP1A1 and/or CYP1B1 gene expression in the human lung cell lines BEP2D and NCIH2009. Fluorescence-HPLC analysis revealed that formation of B[a]P-tetrol-I-1 (hydrolyzed form of the corresponding diol-epoxide) was dependent primarily on CYP1A1. In cells without down-regulation of CYP1A1, the B[a]P-tetrol-I-1 was the major tested isomer formed. In contrast, the B[a]P-cis- and trans-7,8-dihydrodiol isomers were readily formed in cells expressing high levels of either CYP-gene. Simultaneous down-regulation of CYP1A1 and CYP1B1 mRNA resulted in low levels of metabolites overall. Residual unmetabolized B[a]P levels followed the expression of CYP1A1 in an inverse manner. In conclusion, these results indicate a major role of CYP1A1 in the bioactivation of B[a]P to carcinogenic B[a]P-diol-epoxides and in overall metabolism of B[a]P in human lung cell lines. In contrast, both CYP1A1 and CYP1B1 contribute significantly to the formation of the B[a]P-cis- and trans-7,8-dihydrodiol isomers.     

Differential effects of disulfiram and diethyldithiocarbamate on small intestinal and liver microsomal benzo[a]pyrene metabolism

Microsomes isolated from rat small intestinal mucosa and liver were used to study the effects of disulfiram and diethyldithiocarbamate on benzo[a]pyrene monooxygenase activity. This activity was decreased in the intestinal microsomes to 25 per cent of control 24 hr after a single oral dose of disulfiram. In contrast, daily administration of disulfiram for 5 days produced a dose related increase of benzo[a]pyrene monooxygenase activity, above control level. The elevated activities were accompanied by a concomitant increase in the concentration of cytochrome P-450. This benzo[a]pyrene monooxygenase activity was further stimulated by addition of α-naphthoflavone to the incubation medium. Furthermore, the absorption maximum of this cytochrome was at 450 nm in the CO bound reduced difference spectrum. These observations indicate that the disulfiram induced cytochrome P-450 was of the control type. Daily pretreatment with diethyldithiocarbamate impaired both intestinal and liver microsomes at benzo[a]pyrene monooxygenase activities. Pretreatment with a single dose of 3-methylcholanthrene resulted in a more than 10-fold increase of intestinal benzo[a]pyrene monooxygenase activity after 24 hr. Administration of disulfiram 24 hr before treatment appeared to potentiate the 3-methylcholanthrene induced increase of intestinal benzo[a]pyrene monooxygenase activity. In vitro addition of disulfiram and diethyldithiocarbamate to incubates of intestinal or liver microsomes inhibited benzo[a]pyrene metabolism to various extents; the liver being more sensitive. Disulfiram was approximately 50-fold more potent as an inhibitor than diethyldithiocarbamate. The in vitro inhibition of intestinal benzo[a]pyrene monooxygenase activity obtained with disulfiram appeared to be caused both by direct interaction with the monooxygenase system and through NADPH dependent metabolic activation of disulfiram, while the inhibition of diethyldithiocarbamate may be a result of the latter process only.     

On the capacity of human placental enzymes to catalyze the formation of diols from benzo[a]pyrene

Studies were performed on the oxidative biotransformation of benzo[a]pyrene in fortified preparations of human placental microsomes by analysis with high-pressure liquid chromatography. These investigations revealed that the utilization of substrate concentrations (1–2 × 10^?4m) sufficiently high to assure zero-order reaction kinetics (in terms of the generation of phenolic metabolites) produced a marked inhibitory effect on the formation of dihydrodiols in the same reaction mixtures. Relative quantities of dihydrodiols generated increased with decreasing substrate concentrations between 200 and 2.7 μm. Additions of manganese or ferric ions to reaction mixtures altered the ratios of generated phenols to dihydrodiols but did not provide an explanation for the differences observed in the literature. Identical results were obtained with either ¹⁴C- or ³H-labeled benzo[a]pyrene as substrates. The data suggested the possibility that considerable quantities of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, a proximate mutagen/carcinogen, may be generated in vivo by placental tissues of women who smoke.     

Separation of water-soluble metabolites of benzo[a]pyrene formed by cultured human colon.

A method has been developed to separate conjugated metabolites of benzo[a]pyrene into three major fractions: sulfate esters, glucuronides and glutathione conjugates. In cultured human colon, formation of sulfate esters and glutathione conjugates is the major conjugation pathway, while formation of glucuronides accounts for only 6 per cent of the water-soluble metabolites. Hydrolysis of the sulfate esters with arylsulfatase and the glucuronides with β-glucuronidase released metabolites of benzo[a]pyrene that were extractable with organic solvent. Separation of these metabolites by high-pressure liquid chromatography indicated that trans-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene,7,8,9, 10-tetrahydro-7,8,9, 10-tetrahydroxybenzo[a]pyrene and trans-9, 10-dihydro-9, 10-dihydroxybenzo[a]pyrene were the major substrates for UDP-glucuronic acid transferase, while trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene and 9-hydroxybenzo[a]pyrene were the major substrates for sulfotransferase in cultured human colon.     

Hydroxylation of benzo[a]pyrene and binding of (−)trans 5-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene metabolites to deoxyribonucleic acid catalyzed by purified forms of rabbit liver microsomal cytochrome P-450: Effect of 7,8-benzoflavone,butylated hydroxytoluene and ascorbic acid

The catalytic activities of hepatic microsornes from untreated, phenobarbital-treated and 3-methylcholanthrene-treated adult rabbits with respect to benzo[a]pyrene hydroxylation and the activation of (?)(rflw-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene[(?)trans-7,8-diol] to DNA-binding metabolites were determined in the absence and presence of mixed-function oxidase inhibitors and compared to the corresponding activities of the individual enzyme systems. Treatment of rabbits with phnobarbital led to induction of P-450LM₂ and a concomitant 3-fold enhancement in microsomal benzo[a]pyrene hydroxylase activity, whereas the conversion of (?)trans-7,8-diol to DNA-binding products was unaffected. Homogeneous phenobarbital-inducible P-450LM₂ exhibited the highest activity and specificity toward benzo[a]pyrene and the lowest activity toward (?)trans-7,8-diol. Conversely, P-450LM₄ was the major form of cytochrome P-450 induced in rabbit liver by 3-methylcholanthrene or β-naphthoflavone, and this was associated in microsomes with an increase in the metabolism of (?)trans-7, 8-diol but not of benzo[a]pyrene. Homogeneous P-450LM₄ preferentially Catalyzed the oxygénation of (?)trans-7,8-diol, but was largely ineffective with benzo[a]pyrene. Partially purified P-450LM₇ lacked substrate specificity, for it metabolized both benzo[a]pyrene and (?)trans-7, S-diol at comparable rates. Additionally, 7,8-benzoflavone strongly inhibited benzo[a]pyrene hydroxylation by P-450LM₄ and phenobarbital-induced microsomes, as well as (?)trans-7,8-diol metabolism by P-450LM₄ and 3-methyl-cholanthrene-induced microsomes; in contrast, the activity of control microsomes with either substrate, and the activities of P-450LM₄ and LM₂ with benzo[a]pyrene and (?)trans-7 ,8-diol, respectively, were only partially or slightly decreased by 7,8-benzoflavone. Unlike 7,8-benzoflavone, butylated hydroxytoluene inhibited benzo[a]pyrene hydroxylation only. Thus, different forms of rabbit liver microsomal cytochrome P-450 were involved in the metabolism of benzo[a]pyrene and its 7,8-dihydrodiol. The results also demonstrate that the changes in substrate specificity and inhibitor sensitivity seen in phenobarbital- and 3-methylcholanthrene-induced microsomes relative to control rabbit liver microsomes can be accounted for by the catalytic properties of a specific form of cytochrome P-450 that prevails in these preparations, P-450LM₂ and LM₄, respectively.     

Substrate and position specificity of hematin-activated monooxygenation reactions

Rates of hydroxylation of benzo[a]pyrene (BaP), benzo[e]pyrene (BeP), chrysene, acetanilide (AC), 7,12-dimethylbenz[a]anthracene (DMBA), and 17β-estradiol (E₂) in vitro could be increased by as much as 70-fold by additions of micromolar quantities of hematin. Such increases were observed primarily when extrahepatic tissues were utilized as the enzyme source; the greatest increases occurred with rabbit brain. Mixed-function oxygenations of aniline, ethylmorphine, benzphetamine, N-2-fluorenylacetamide (FAA), dibenz[a, h] anthracene(DBA), and benz[a] anthracene (BA), were affected minimally or not at all by hematin additions. Analyses of metabolites of BaP, AC and DMBA with high-pressure liquid chromatography revealed a high degree of position specificity for the hematinmediated reactions. This specificity was dependent upon the enzyme source, e.g. with rabbit kidney as enzyme source and AC as substrate, hematin additions resulted in only minor increases (30–40 per cent) in quantities of 3- and 4-hydroxylated metabolites and decreases (approximately 40–50 per cent; possibly a result of further degradation) in amounts of 2-hydroxylated AC. With hematin additions to rabbit brain homogenates, quantities of the measured 2-hydroxylated AC increased by 10 to 12-fold and of the 3-hydroxylated product by 3 to 4-fold, but no detectable changes in 4-hydroxylated AC were observed. With BaP as substrate, hematin elicited the formation of large quantities of an unidentified and hitherto undetected metabolite. Results of the study were consistent with the concept that hematin additions result in the reconstitution of a number of functionally distinct, tissue-specific cytochrome P-450 apoproteins.     

Discrimination of a transformation phenotype in Syrian golden hamster embryo (SHE) cells using ATR-FTIR spectroscopy

Primary Syrian hamster embryo (SHE) cells might be used to assess morphological transformation following treatment with chemical carcinogens. We employed attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy to interrogate SHE colonies, as complex biomolecules absorb in the mid-infrared (IR; λ = 2–20 μm) giving vibrational spectra associated with structure and function. Early-passage SHE cells were cultured (pH 6.7) in the presence or absence of benzo[a]pyrene (B[a]P; 5.0 μg/ml). Unstained colonies were applied to an ATR crystal, and vibrational spectra were obtained in the ATR mode using a Bruker Vector 22 FTIR spectrometer with Helios ATR attachment. These were individually baseline-corrected and normalised. Spectra were then analysed using principal component analysis (PCA) plus linear discriminant analysis (LDA). PCA was used to reduce the dataset dimensions before LDA was employed to reveal clustering. This determined whether wavenumber–absorbance relationships expressed as single points (scores) in ‘hyperspace’ might on the basis of multivariate distance reveal biophysical differences associated with morphologies in vehicle control (non-transformed or transformed) or carcinogen-treated (non-transformed or transformed) cells. Retrospectively designated SHE colonies (following staining and microscopic analysis) clustered according to whether they were vehicle control (non-transformed), B[a]P-treated (non-transformed) or transformed (control and B[a]P-treated). Scores plots pointed to a B[a]P-treated phenotype and derived loadings plots highlighted distinguishing markers in control transformed vs. B[a]P-treated transformed; these were mostly associated with Amide I, Amide II and phosphate stretching (asymmetric and symmetric) vibrations. Combined application of ATR-FTIR spectroscopy and unsupervised (PCA)/supervised (LDA) may be a novel approach to scoring SHE colonies for morphological transformation.