Epimerization of benzo[a]pyrene-tetrols after acid hydrolysis, implications for determination of benzo[a]pyrene adducts in protein and DNA

Determination of benzo[a]pyrene-DNA or protein adducts with high performance liquid chromatography (HPLC) after acid hydrolysis at high temperature (90 degrees C) enables four isomers of benzo[a]pyrene tetrahydrotetrol to be identified and quantitated. We have investigated the effect of acid treatment of benzo[a]pyrene-tetrahydrotetrol isomers using HPLC and nuclear magnetic resonance spectroscopy (NMR) analysis. By HPLC, we found reversible epimerization of (+/-)-benzo[a]pyrene-r-7,t-8,9, 10-tetrahydrotetrol to (+/-)-benzo[a]pyrene-r-7,t-8,9, c-10-tetrahydrotetrol and of (+/-)-benzo[a]pyrene-r-7,t-8,c-9, t-10-tetrahydrotetrol to (+/-)-benzo[a]pyrene-r-7,t-8,c-9, 10-tetrahydrotetrol, but no interconversion between the two isomer groups. After acid hydrolysis, we found an equilibrium of 87% (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol and 9% (+/-)-benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol and 68% (+/-)-benzo[a]pyrene-r-7,t-8,c-9,10-tetrahydrotetrol and 20% (+/-)-benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol. Minor amounts of two unknown compounds with similar chromatographic characteristics were also found. We have established a NMR method for determination of underivatized (+/-)-benzo[a]pyrene-r-7,t-8,9, c-10-tetrahydrotetrol and (+/-)-benzo[a]pyrene-r-7,t-8,9, 10-tetrahydrotetrol confirming the epimerization of (+/-)-benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol to (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10- tetrahydrotetrol. (+/-)-Benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol was treated with aqueous hydrochloric acid in tetrahydro- furan-d8 to give (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol at 57 degrees C while observing the 1H NMR resonances at 500 MHz. Gradient-selected correlation spectroscopy (COSY), heteronuclear multiple quantum correlation (HMQC) and heteronuclear multiple bond correlation (HMBC) experiments were performed to confirm the assignments of the aliphatic hydrogens in the product (+/-)-benzo[a]pyrene-r-7,t-8,9, c-10-terahydrotetrol. Thus, when analyzing benzo[a]pyrene-DNA or protein adducts by cleaving the adducts with acid hydrolysis, the only ratio of biological significance is between (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol plus (+/-)-benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol and (+/-)-benzo[a]pyrene-r-7,t-8,c-9,10-tetrahydrotetrol plus (+/-)-benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol, due to interconversion (epimerization) at C-10.     

Two-step error-prone bypass of the (+)- and (−)-trans-anti-BPDE-N-dG adducts by human DNA polymerases η and κ

Benzo[a]pyrene is a polycyclic aromatic hydrocarbon (PAH) associated with potent carcinogenic activity. Mutagenesis induced by benzo[a]pyrene DNA adducts is believed to involve error-prone translesion synthesis opposite the lesion. However, the DNA polymerase involved in this process has not been clearly defined in eukaryotes. Here, we provide biochemical evidence suggesting a role for DNA polymerase η (Polη) in mutagenesis induced by benzo[a]pyrene DNA adducts in cells. Purified human Polη predominantly inserted an A opposite a template (+)- and (−)-trans-anti-BPDE-N²-dG, two important DNA adducts of benzo[a]pyrene. Both lesions also dramatically elevated G and T mis-insertion error rates of human Polη. Error-prone nucleotide insertion by human Polη was more efficient opposite the (+)-trans-anti-BPDE-N²-dG adduct than opposite the (−)-trans-anti-BPDE-N²-dG. However, translesion synthesis by human Polη largely stopped opposite the lesion and at one nucleotide downstream of the lesion (+1 extension). The limited extension synthesis of human Polη from opposite the lesion was strongly affected by the stereochemistry of the trans-anti-BPDE-N²-dG adducts, the nucleotide opposite the lesion, and the sequence context 5′ to the lesion. By combining the nucleotide insertion activity of human Polη and the extension synthesis activity of human Polκ, effective error-prone lesion bypass was achieved in vitro in response to the (+)- and (−)-trans-anti-BPDE-N²-dG DNA adducts.     

HPLC analysis of benzo[a]pyrene-albumin adducts in benzo[a]pyrene exposed rats. Detection of cis-tetrols arising from hydrolysis of adducts of anti- and syn-BPDE-III with proteins

Quantitation of protein-benzo[a]pyrene adducts represent a more sensitive analysis method than quantitation of benzo[a]pyrene-DNA adducts. By accurate analysis of benzo[a]pyrene-protein adducts several different molecular adduct forms can be studied. Male Wistar rats were injected i.p. with benzo[a]pyrene, and serum albumin was isolated and subjected to acid hydrolysis at 90 degrees C for 3 h. The hydrolysate was analyzed by HPLC with fluorescence detection. The HPLC profiles obtained after albumin hydrolysis from benzo[a]pyrene exposed animals were compared to similar HPLC profiles from in vitro adducted bovine serum albumin (BSA) and direct hydrolysis of both r-10,t-9-dihydrodiol-c-7,8-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (syn-BPDE-III) and r-10,t-9-t-dihydrodiol-t-7,8-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE-III). After acid hydrolysis of albumin from benzo[a]pyrene exposed rats, 6 fluorescent peaks were separated. Four of the peaks were isomers of benzo[a]pyrene-tetrahydrotetrols, (+/-)-benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol, (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol, (+/-)-benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol and (+/-)-benzo[a]pyrene-r-7,t-8,c-9,10-tetrahydrotetrol. In addition we found two fluorescent peaks, named X1 and X2 with retention times similar to the benzo[a]pyrene-tetrols. The unknown fluorescent peaks reacted similar to the four known tetrols in both dose response experiments and time course experiments. Fluorescent material with retention times equal to X1 and X2 were found after acid hydrolysis of syn-BPDE-III and anti-BPDE-III in acid and in hydrolysates from BSA treated in vitro with syn-BPDE-III and anti-BPDE-III. The ratio X1/X2 was relatively constant indicating epimerization equilibrium between these to species. Synchronous fluorescence analysis of fractions containing X1 or X2 from both in vivo and in vitro experiments showed fluorescence spectra characteristic of benzo[a]pyrene tetrols using a wavelength difference of 34 nm.     

Determinants of anti-benzo[a]pyrene diol epoxide–DNA adduct formation in lymphomonocytes of the general population

We evaluated determinants of anti-benzo[a]pyrenediolepoxide-(B[a]PDE)–DNA adduct formation (adduct induced by the ultimate carcinogenic metabolite of B[a]P) in lymphomonocytes of subjects environmentally exposed to low doses of polycyclic aromatic hydrocarbons (PAHs) (B[a]P). Our study population consisted of 585 Caucasian subjects, all municipal workers living in North-East Italy and recruited during their periodic check-ups after informed consent. PAH (B[a]P) exposure was assessed by questionnaire. Anti-B[a]PDE–DNA levels were measured by HPLC fluorescence analysis.We found that cigarette smoking (smokers (22%) versus non-smokers, p < 0.0001), dietary intake of PAH-rich meals (≥52 (38%) versus <52 times/year, p < 0.0001), and outdoor exposure (≥4 (19%) versus <4 h/day; p = 0.0115) significantly influenced adduct levels. Indoor exposure significantly increased the frequency of positive subjects (≥0.5 adducts/10⁸ nucleotides; χ² for linear trend, p = 0.051). In linear multiple regression analysis the major determinants of increased DNA adduct levels (ln values) were smoking (t = 6.362, p < 0.0001) and diet (t = 4.035, p < 0.0001). In this statistical analysis, indoor and outdoor exposure like other factors of PAH exposure had no influence. In non-smokers, the influence of diet (p < 0.0001) and high indoor exposure (p = 0.016) on anti-B[a]PDE–DNA adduct formation became more evident, but not that of outdoor exposure, as was confirmed by linear multiple regression analysis (diet, t = 3.997, p < 0.0001 and high indoor exposure, t = 2.522, p = 0.012).This study indicates that anti-B[a]PDE–DNA adducts can be detected in the general population and are modulated by PAH (B[a]P) exposure not only with smoking – information already known from studies with limited number of subjects – but also with dietary habits and high indoor exposure. In non-smokers, these two factors are the principal determinants of DNA adduct formation. The information provided here seems to be important, since DNA adduct formation in surrogate tissue is an index of genotoxic exposure also in target organs (e.g., lung) and their increase may also be predictive of higher risk for PAH-related cancers.     

Unexpected DNA damage caused by polycyclic aromatic hydrocarbons under standard laboratory conditions

The genotoxicity of 15 polycyclic aromatic hydrocarbons was determined with the alkaline version of the comet assay employing V79 lung fibroblasts of the Chinese hamster as target cells. These cells lack the enzymes necessary to convert PAHs to DNA-binding metabolites. Surprisingly, 11 PAHs, i.e., benzo[a]pyrene (BaP), benz[a]anthracene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene, fluoranthene, anthanthrene, 11H-benzo[b]fluorene, dibenz[a,h]anthracene, pyrene, benzo[ghi]perylene and benzo[e]pyrene caused DNA strand breaks even without external metabolic activation, while naphthalene, anthracene, phenanthrene and naphthacene were inactive. When the comet assay was performed in the dark or when yellow fluorescent lamps were used for illumination the DNA-damaging effect of the 11 PAHs disappeared. White fluorescent lamps exhibit emission maxima at 334.1, 365.0, 404.7, and 435.8 nm representing spectral lines of mercury. In the case of yellow fluorescent lamps these emissions were absent. Obviously, under standard laboratory illumination many PAHs are photo-activated, resulting in DNA-damaging species. This feature of PAHs should be taken into account when these compounds are employed for the initiation of skin cancer.The genotoxicity of BaP that is metabolically activated in V79 cells stably expressing human cytochrome P450-dependent monooxygenase (CYP1A1) as well as human epoxide hydrolase (V79-hCYP1A1-mEH) could not be detected with the comet assay performed under yellow light. Likewise the DNA-damaging effect of r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BaPDE) observed with the comet assay was only weak. However, upon inhibition of nucleotide excision repair (NER), which is responsible for the removal of stable DNA adducts caused by anti-BaPDE, the tail moment rose 3.4-fold in the case of BaP and 12.9-fold in the case of anti-BaPDE. These results indicate that the genotoxicity of BaP and probably of other compounds producing stable DNA adducts are reliably detected with the comet assay only when NER is inhibited.     

Inhibition of the benzo[<Emphasis Type="Italic">a</Emphasis>]pyrene-induced toxicity by allyl sulfides in human epidermal keratinocytes

Diallyl sulfide (DAS) and diallyl disulfide (DADS) at 25 g ml^–1decreased the benzo[a]pyrene (B[a]P)-induced colony growth inhibition of human epidermal keratinocytes. DAS and DADS decreased B[a]P-DNA and B[a]P-protein adducts by 65% and 49–55%, respectively. The B[a]P-induced ethoxyresorufin O-deethylase activity, a marker enzyme for cytochrome P450 1, was decreased from 3 to 1.7–1.9 nmol min^–1 mg^–1 microsomal protein by DAS and DADS treatments. The activity of glutathione S-transferase, a detoxifying enzyme for B[a]P, but was decreased by DADS, but was unaffected by DAS.     

P-Postlabeling high-performance liquid chromatographic improvements to characterize DNA adduct stereoisomers from benzo[a]pyrene and benzo[c]phenanthrene, and to separate DNA adducts from 7,12-dimethylbenz[a]anthracene

Single compounds can generate complex DNA adduct patterns by reactions through different pathways, with different target nucleotides and through different configurations of the products. DNA adduct analysis by ³²P-HPLC was improved by adding an isocratic plateau in an otherwise linear gradient, thereby enhancing resolution of predictable retention time intervals. This enhanced ³²P-HPLC technique was used to analyze and at least partly resolve 14 out of 16 available benzo[c]phenanthrene deoxyadenosine and deoxyguanosine adduct standards, 8 out of 8 available benzo[a]pyrene deoxyadenosine and deoxyguanosine adduct standards, and 51 peaks from 7,12-dimethylbenz[a]anthracene-calf thymus DNA reaction products. The same type of gradient modifications could be used to enhance resolution in analyses of other complex DNA adduct mixtures, e.g., in vivo in humans.     

Assessment of occupational exposure to PAHs in an Estonian coke oven plant- correlation of total external exposure to internal dose measured as 1-hydroxypyrene concentration

The exposure of cokery workers to polynuclear aromatic hydrocarbons at an Estonian oil shale processing plant was assessed by using occupational hygiene and biomonitoring measurements which were carried out twice, in midwinter and in the autumn. To assess the external dose of polynuclear aromatic hydrocarbons, pyrene and benzo[a]pyrene concentrations were measured from the breathing zone of workers during a workshift. Skin contamination with pyrene and benzo[a]pyrene was assessed by skin wipe sampling before and after the workshift. As a biomarker of overall exposure to polynuclear aromatic hydrocarbons, and as an integral of all absorption routes of pyrene, 1-hydroxypyrene concentration was measured from post shift urine samples. Of the personal air samples, 18% exceeded the Finnish threshold limit value of benzo[a]pyrene (10 μg m^-3). Mean value (two separate measurements together) for benzo[a]pyrene was 5.7 μg m^-3 and for pyrene, 8.1 μg m^-3. Based on skin wipe sample analyses, the skin contamination was also obvious. The mean value of benzo[a]pyrene in the samples collected after the shift was 1.2 ng cm^-2. Benzo[a]pyrene was not found in control samples. The mean value of urinary 1-hydroxypyrene concentration was 6.0 μmol mol^-1 creatinine for the exposed workers and 0.5 μmol mol^-1 creatinine for the controls. This study undoubtedly shows the usefulness of 1-hydroxypyrene as an indicator of internal dose of polynuclear aromatic hydrocarbons. It can be concluded that the cokery workers at the Kohtla-Järve plant are exposed to high concentrations of polynuclear aromatic compounds, and the exposure level is considerably higher during the winter measurements.     

Rat liver homogenate (S9)-mediated binding of benzo[a]pyren to DNA in V79 cells,V79 cell nuclei and aqueous solution

The role of the target cell in determining the structures and the amounts of hydrocarbon-DNA adducts formed after hydrocarbon activation by an exogenous metabolic ativation system was investigated by exposing intact cells of the Chinese hamster lung cell line V79, V79 cell nuclei and calf thymus DNA to benzo[a]pyrene (B[a]P) in the presenceof a rat liver homogenate activation system (S9). The DNA was isolated, enzymatically degraded to deoxyribonucleosides and the B[a]P-deoxyribonucleoside adducts analyzed by high-performance liquid chromatography. Two major adducts were present in all samples; one formed by reaction of r-7, t-8-dihydroxy-t-9, 10-epoxy-7, 8, 9, 10-tetrahydro-B[a]P (anti-B[a]PDE) with the 2-amino group of deoxyguanosine, the other formed by reaction of a metabolite of 9-hydroxybenzo[a]pyrene (9-OH-B[a]P) with an unidentified deoxyribonucleoside. The ratios of the anti-B[a]PDE-DNA adduct to the 9-OH-B[a]P-DNA adduct were: calf thymus DNA, 3 to 1: DNA from V79 nuclei, 8 to 1; DNA from intact V79 cells, 11 to 1. Similar several-fold increases in the proportion of anti-B[a]PDE-DNA adducts in V79 cells over those in calf thymus DNA were observed for a dose range of 1–10 μg B[a]P per ml. The relative extent of binding of the activated metabolite of 9-OH-B[a]P to DNA was also much lower in intact V79 cells than in calf thymus DNA after exposure to 9-OH-B[a]P in the presence of the S9 activation system.These results demonstrate that the relative abilities of various reactive bbenzo[a]pyrene metabolites formed by an exogenous activation system to reach DNA differ substantially. Therefore, assessment of the biological activity of hydrocarbons in mutation assays using exogenous activation systems must take into account not only the amounts of different reactive hydrocarbon metabolites formed but also the relative abilities of these metabolites to reach the DNA of the target cell.     

Time course of metabolism of benzo[e]pyrene by hamster embryo cells and the effect of chemical modifiers

In cultures of hamster embryo cells, benzo[a]pyrene (B[a]P) is metabolized primarily in the bay region. In contrast, little or no bay region metabolism of the noncarcinogenic isomer benzo[e]pyrene (B[e]P) could be detected during 12–96-h incubations of hamster embryo cells with 4 μM [³H]B[e]P. The upper limit to 9,10-dihydro-9,10-dihydroxy-B[e]P formation is about 0.2% of the ethyl acetate-soluble metabolites ( <0.1% of the total metabolites). The major identified metabolites of B[e]P were 4,5-dihydro-4,5-dihydroxy B[e]P and the glucuronide conjugates of 3-OH-B[e]P and 4,5-dihydro-4,5-dihydroxy B[e]P. Simultaneous treatment of cells with either B[a]P or 7,8-benzoflavone (BF) did not induce bay region metabolism of [³H]B[e]P.     

Degradation of eight highly condensed polycyclic aromatic hydrocarbons by Pleurotus sp. Florida in solid wheat straw substrate

The degradation of eight unlabeled highly condensed polycyclic aromatic hydrocarbons (PAH) and the mineralization of three ¹⁴C-labeled PAH by the white-rot fungus Pleurotus sp. Florida was investigated. Three concentrations containing 50, 250 or 1250 μg each unlabeled PAH/5 g straw were added to sterile sea sand. Selected treatments were added subsequently with ¹⁴C-labeled pyrene, benzo[a]anthracene or benzo[a]pyrene. The PAH-loaded sea sand was then mixed into straw substrate and incubated. The disappearance of the unlabeled four-to six-ring PAH: pyrene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenz[a,h]anthracene and benzo[ghi]perylene, was determined by high-performance liquid chromatography. After 15 weeks of incubation, the recoveries were less than 25% for initial amounts of 50 μg (controls above 85%). The recoveries of unlabeled PAH increased in the inoculated samples with increasing concentrations applied. No correlation could be determined between the number of condensed rings of the PAH and the recoveries of added PAH. Pleurotus sp. Florida mineralized 53% [¹⁴C]pyrene, 25% [¹⁴C]benzo[a]anthracene and 39% [¹⁴C]benzo[a]pyrene to ¹⁴CO₂ in the presence of eight unlabeled PAH (50 μg applied) within 15 weeks. During the course of cultivation, Pleurotus sp. Florida degraded more than 40% of the wheat straw substrate. Variation of the initial concentration of PAH did not influence the extent of degradation of the organic matter. Received: 16 December 1996 / Received revision: 17 March 1997 / Accepted: 22 March 1997     

Trace determination of urinary 3-hydroxybenzo[a]pyrene by automated column-switching high-performance liquid chromatography

3-Hydroxybenzo[a]pyrene (3-OHB[a]P), one of the metabolites of benzo[a]pyrene (B[a]P), has been determined in human urine using an automated column-switching procedure. The hydrolysed biological sample is centrifuged just prior to being injected into a reusable precolumn loop, which is packed with a preparative phase and coupled on-line to a liquid chromatographic (LC) system. A rapid pre-treatment of the hydrolysed sample, consisting of a concentration and a crude clean-up, is performed on the precolumn. The analytes are then non-selectively desorbed with the LC eluent and the sample is cleaned again in three successive purification columns using the direct transfer or “heart-cut” technique. The pre-treatment does not exceed 3 min. and the entire analytical purification and separation procedure takes less than 30 min. Average 3-OHB[a]P recovery reaches 95% in the 1–50 ng/l range of urine, and the detection limit is 0.1 ng/l urine for a 3 ml injection of hydrolysed urine. The developed method was compared with a more time-consuming off-line method to analyse urines of B[a]P gavaged rats; the statistical treatment indicates that both methods are in agreement. The method was applied to purify and concentrate the urine samples of workers exposed and apparently unexposed to polycyclic aromatic hydrocarbons (PAHs).     

Benzo{a}pyrene-induced DNA damage in Mytilus galloprovincialis: measurement of bulky DNA adducts and DNA oxidative damage in terms of 8-oxo-7,8-dihydro-2´-deoxyguanosine formation

Bulky DNA adducts and 8-oxo-7,8-dihydro-2´-deoxyguanosine (8-oxodGuo) were measured in gill DNA of benzo[a]pyrene (B[a]P)-exposed mussels (50 mg kg^-1 dw day^-1), respectively by the ³²P-post-labelling technique and high performance liquid chromatography coupled to electrochemical detection assay. A time-course study was performed for both biomarkers and their potential use for marine biomonitoring discussed for the sentinel species studied. In gills, B[a]P-related DNA adducts were positively correlated with B[a     

Metabolite repression inhibits degradation of benzo[a]pyrene and dibenz[a,h]anthracene by Stenotrophomonas maltophilia VUN 10,003

Large inocula of Stenotrophomonas maltophilia VUN 10,003 were used to investigate bacterial degradation of benzo[a]pyrene and dibenz[a,h]anthracene. Although strain VUN 10,003 was capable of degrading 10–15 mg l⁻¹ of the five-ring compounds in the presence of pyrene after 63 days, further addition of pyrene after degradation of the five-ring polycyclic aromatic hydrocarbons (PAHs) ceased did not stimulate significant decreases in the concentration of benzo[a]pyrene or dibenz[a,h]anthracene. However, pyrene was degraded to undetectable levels 21 days after its addition. The amount of benzo[a]pyrene and dibenz[a,h]anthracene degraded by strain VUN 10,003 was not affected by the initial concentration of the compounds when tested at 25–100 mg l⁻¹, by the accumulation of by-products from pyrene catabolism or a loss of ability by the cells to catabolise benzo[a]pyrene or dibenz[a,h]anthracene. Metabolite or by-product repression was suspected to be responsible for the inhibition: By-products from the degradation of the five-ring compounds inhibited their further degradation. Journal of Industrial Microbiology & Biotechnology (2002) 28, 88–96 DOI: 10.1038/sj/jim/7000216 Received 30 January 2001/ Accepted in revised form 10 October 2001     

Degradation of benz[<Emphasis Type="Italic">a</Emphasis>]anthracene by <Emphasis Type="Italic">Mycobacterium vanbaalenii</Emphasis> strain PYR-1

Cultures of Mycobacterium vanbaalenii strain PYR-1 grown in mineral salts medium and nutrients in the presence of benz[a]anthracene metabolized 15% of the added benz[a]anthracene after 12days of incubation. Neutral and acidic ethyl acetate extractable metabolites were isolated and characterized by high performance liquid chromatography (HPLC) and uv–visible absorption, gas chromatography/mass (GC/MS) and nuclear magnetic resonance (NMR) spectral analysis. Trimethylsilylation of the metabolitesfollowed by GC/MS analysis facilitated identification of metabolites. The characterization of metabolites indicated that M. vanbaalenii initiated attack of benz[a]anthracene at the C-1,2-, C-5,6-, C-7,12- and C-10,11-positions to form dihydroxylated and methoxylated intermediates. The major site of enzymatic attack was in the C-10, C-11 positions. Subsequent ortho- and meta-cleavage of each of the aromatic rings led to the accumulation of novel ring-fission metabolites in the medium. The major metabolites identified were 3-hydrobenzo[f]isobenzofuran-1-one (3.2%), 6-hydrofuran[3,4-g]chromene-2,8-dione (1.3%), benzo[g]chromene-2-one (1.7%), naphtho[2,1-g]chromen-10-one (48.1%), 10-hydroxy-11-methoxybenz[a]anthracene (9.3%), and 10,11-dimethoxybenz[a]anthracene (36.4%). Enzymatic attack at the C-7 and C-12 positions resulted in the formation of benz[a]anthracene-7,12-dione, 1-(2-hydroxybenzoyl)-2-naphthoic acid, and 1-benzoyl-2-naphthoic acid. A phenyl-naphthyl metabolite, 3-(2-carboxylphenyl)-2-naphthoic acid, was formed when M. vanbaalenii was incubated with benz[a]anthracene cis-5,6-dihydrodiol, indicating ortho-cleavage of 5,6-dihydroxybenz[a]anthracene. A minor amount of 5,6-dimethoxybenz[a]anthracene was also formed. The data extend and propose novel pathways for the bacterial metabolism of benz[a]anthracene.     

Rutaceous alkaloids as models for the design of novel antitumor drugs

The chemical diversity of alkaloids in the Rutaceae is correlated with biosynthetic pathways involving various aromatic amino acid precursors, tyrosine, tryptophan, histidine, and anthranilic acid. The interest of rutaceous polyheteroaromatic alkaloids as models for the development of anticancer agents relies on their frequent ability to interact with DNA or with systems involved in the control of its topology, repair, and replication. Fagaronine and nitidine, from Zanthoxylum, demonstrate antileukemic activity, associated with topoisomerases inhibition. Evodiamine from Euodia rutaecarpa, displays antimetastatic properties. The pyranoacridone acronycine, from Sarcomelicope, exhibits antitumor activity against a broad spectrum of solid tumors. Development of synthetic analogues based on this latter natural product template followed the isolation of the unstable acronycine epoxide, which led to a hypothesis of bioactivation of acronycine by transformation of the 1,2-double bond into the corresponding oxirane. 1,2-Diacyloxy-1,2-dihydroacronycine derivatives exhibited antitumor properties, with a broadened spectrum of activity and an increased potency. The demonstration that acronycine interacted with DNA led to develop benzo[a], [b], and [c]acronycine analogs. Benzo[a] and [b] derivatives displayed significant antitumor activities. 1,2-Dihydroxy-1,2-dihydrobenzo[b]acronycine esters and diesters were active in human orthotopic models of cancers xenografted in nude mice. The activity of these compounds was correlated with their ability to give covalent adducts with DNA, involving reaction between the N-2 amino group of guanines and the ester group at the benzylic position of the drug. Cis-1,2-diacetoxy-1,2-dihydrobenzo[b]acronycine, currently developed under the code S23906-1, successfully underwent phase I and is currently under phase II clinical trials.     

Synthesis of carbon-11-labeled CK1 inhibitors as new potential PET radiotracers for imaging of Alzheimer’s disease

The reference standards methyl 3-((2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)carbamoyl)benzoate (5a) and N-(2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)-3-methoxybenzamide (5c), and their corresponding desmethylated precursors 3-((2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)carbamoyl)benzoic acid (6a) and N-(2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)-3-hydroxybenzamide (6b), were synthesized from 5-amino-2,2-difluoro-1,3-benzodioxole and 3-substituted benzoic acids in 5 and 6 steps with 33% and 11%, 30% and 7% overall chemical yield, respectively. Carbon-11-labeled casein kinase 1 (CK1) inhibitors, [¹¹C]methyl 3-((2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)carbamoyl)benzoate ([¹¹C]5a) and N-(2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)-3-[¹¹C]methoxybenzamide ([¹¹C]5c), were prepared from their O-desmethylated precursor 6a or 6b with [¹¹C]CH₃OTf through O-[¹¹C]methylation and isolated by HPLC combined with SPE in 40–45% radiochemical yield, based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB). The radiochemical purity was >99%, and the molar activity (MA) at EOB was 370–740?GBq/μmol with a total synthesis time of ～40-min from EOB.     

Microsomal metabolism of dibenz[a,c]anthracene, dibenz[a,h]anthracene and dibenz[a,j]anthracene to bis-dihydrodiols and polyhydroxylated products

Polar, ethyl acetate soluble metabolites formed in incubations of dibenz[a,c]-anthracene (DB[a,c]A), dibenz[a,h]anthracene (DB[a,h]A) and the related DB[a,h]A 3,4-diol and dibenz[a,j]anthracene (DB[a,j]A) with 3-methylcholanthrene (3-MC)-induced rat liver microsomal preparations have been separated by HPLC and examined using fluorescence, UV and NMR spectroscopy. Metabolites with spectral properties consistant with their identification as the 3,4:8,9-bis-diol of DB[a,j]A and a 1,2,3,4,12,13-hexol derived from DB[a,c]A were found. DB[a,h]A was metabolized to three polar products identified as the 3,4:10,11-bis-diol and the related 1,2,3,4,8,9- and 1,2,3,4,10,11-hexols, which were also formed, together with the related 1,2,3,4-tetrol, from the DB[a,h]A 3,4-diol. The possible role of bis-diols in the metabolic activation of these three dibenzanthracenes is discussed.     

Selection of a support for immobilization of a microbial lipase for the hydrolysis of triglycerides

Baterial lipase from Staphylococcus carnosus (pLipMut2) has been immobilized on various supports in order to determine a suitable immobilization technique in terms of activity and stability, when utilized for the hydrolysis of tributyrin. The hydrophobic materials PBA Eupergit and PBA Eupergit 250L prooved to be appropriate supports, when the enzyme was crosslinked with glutaraldehyde after adsorption. No desorption of the immobilized enzyme occured during operation. The pore size of the support has a strong effect on the activity but does not influence stability.The initial activity for immobilized and soluble lipase is found to follow the Arrhenius equation at low temperature, where mass transfer does not affect reaction kinetics. Activation energies for soluble and immobilized lipase were evaluated to be 21.7 kJ mol^–1 and 60.8 kJ mol^–1, respectively.Operational stability was studied in a packed bed recirculation reactor. Thermal desactivation followed first order kinetics with a half-life of 1340 h at 10°C. Model calculations for productivity showed, that optimal temperatures for high productivity are well below the temperature of maximal activity.List of Symbols E _a [kJ mol^–1] activation energy - E _d [kJ mol^–1] activation energy of desactivation - H [–] half-number - k _d [h^–1] desactivation constant - k _d, [h^–1] constant - k _N [–] desactivation constant (number) - N [–] number of runs - p [mol dm^–3] productivity - t [h] time - t _0.5 [h] half-life - T [K] absolute temperature - V [U ml^–1] activity - V(N) [Uml^–1] activity exhibited in the n-th run - V _s,O [U ml^–1] initial activity of supernatant - V _s, [U ml^–1] activity of supernatant after immobilization - V _O [U ml^–1] initial activity - V [U ml^–1] constant - _imm [–] activity yield - [ml ml^–1] ratio of volume of support to volume of supernatant Financial support of this work by the Deutsche Forschungsgemeinschaft (SFB 145, A15) is gratefully acknowledged.     

Kinetics of reextraction of Penicillin G from the organic phase after reactive extraction

The kinetics of reextraction of penicillin from the LA-2-penicillin complex-n-butylacetate solution by phosphate buffer was investigated experimentally and theoretically. The concentration time functions can be described by a mathematical model in a wide range of pH-values and complex concentrations.List of Symbols a _P [m²/m³] specific interfacial area - c _A , c _Ai [mMol] concentration of amine in the bulk of the organic phase and at the interface - c _AHP , c _AHPi [mMol] concentration of the complex in bulk of the organic phase and at the interface - c _H , c _Hi [mMol] concentration of the proton in the bulk of the aqueous phase and at the interface - c _P , c _Pi [mMol] concentration of the penicillin acid anion in the bulk of the aqueous phase and at the interface - J _A , J _AHP , J _P [mMol · cm · s^–1] fluxes of amine, complex and penicillin acid anion - k _A , k _AHP , k _P [cm · s^–1] mass transfer coefficients of amine, complex and penicillin acid anion - K _G [mMol²] equilibrium constant - t [k] extraction time