Rabbit alveolar macrophage-mediated mutagenesis of polycyclic aromatic hydrocarbons in V79 Chinese hamster cells

Rabbit pulmonary alveolar macrophages (PAM) were used as a metabolizing device in combination with V79 Chinese hamster cells as a mutational indicator system. The capacity for metabolic activation of benzo[a]pyrene (B[a]P), its 7,8-diol and 2-aminoanthracene by PAM was investigated. Because of the high variation between different PAM preparations, a statistically significant effect of the 3 compounds could only be demonstrated in a series of 4 or 5 experiments. When the ability of PAM to metabolize B[a]P and the 7,8-diol to mutagenic products was compared with that of primary embryonic fibroblasts from Syrian hamsters, PAM were found to be one-tenth as efficient. Experiments were performed to find out how the phagocytic process could affect the metabolic activation of polycyclic aromatic hydrocarbons. The results showed that PAM-mediated mutagenesis of the 7,8-diol was enhanced 5-10-fold if PAM were fed with opsonized particles. The mechanism by which the phagocytosis of PAM enhanced the mutagenicity of 7,8-diol, as detected in co-cultivated V79 cells, can so far only be a matter for speculation.     

Biomonitoring of polycyclic aromatic hydrocarbons in human urine

Measurement of polycyclic aromatic hydrocarbons (PAH) metabolites in human urine is the method of choice to determine occupational and/or environmental exposure of an individual to PAH, in particular, when multiple routes of exposure have to be taken into account. Requirements for methods of biomonitoring PAH metabolites in urine are presented. Studies using 1-hydroxypyrene or phenanthrene metabolites including its phenols and dihydrodiols are summarized. The role of these PAH metabolites as established biomarkers and also more recent developments of PAH biomonitoring are discussed.     

Chromosome aberrations in cultured central mudminnow heart cells and Chinese hamster ovary cells exposed to polycyclic aromatic hydrocarbons and sediment extracts

1. Genotoxicity experiments were conducted with cultured fish cells to determine if the high frequency of epidermal papillomas observed in lemon sole from Sturgeon Bank, where a sewage treatment plant discharges, could be correlated with contamination of the sediments with chemicals such as 3,4-benzopyrene. 2. The frequency of chromosome aberrations was measured in cultured Umbra limi heart (U1-H) and Chinese hamster ovary (CHO) cells following exposure to the polycyclic aromatic hydrocarbons (PAH) 3,4-benzopyrene (BP), 1,2,5,6-dibenzanthracene (DBA), 1,2-benzanthracene (BA), and pyrene (PY), activated using S9 prepared from rainbow trout liver. 3. An increase in the chromosome aberration frequency was only observed following exposure to fish S9-activated BP in both cell lines. 4. Following exposure of the cells to both Sturgeon Bank and Spanish Bank sediment extracts, it was determined that a higher level of toxic and genotoxic activity was associated with the Sturgeon Bank sediments. 5. Since the detection of PAH genotoxicity requires the presence of S9, and since a higher level of genotoxic activity was noted following sediment extract exposures with no S9 present, this suggests that the extracts contain a complex mix of chemicals, some of which express genotoxic activity. 6. An assessment using the micronucleus test failed to indicate in vivo genotoxicity in fish collected from Sturgeon and Spanish Banks. 7. It was, therefore, difficult to associate the observed sediment genotoxicity with the previously noted high incidence of epidermal papillomas in lemon sole from this area.     

Mechanism and rate of permeation of cells by polycyclic aromatic hydrocarbons

The principal mechanism of cellular uptake of benzo(a)pyrene and other polycyclic aromatic hydrocarbons (PAH) from lipoproteins into cells is spontaneous transfer through the aqueous phase (Plant, A. L., Benson, D.M., and Smith, L.C. (1985) J. Cell Biol. 100, 1295-1308). Cellular uptake of benzo(a)pyrene from low density lipoproteins followed first-order kinetics with a rate constant that was independent of the relative lipoprotein concentrations or cell number but which was 2 orders of magnitude smaller than the rate constant for benzo(a)pyrene desorption from low density lipoproteins. Moreover, identical rate constants for cellular uptake of benzo(a)pyrene were observed when the donor vehicle was high density lipoproteins, very low density lipoproteins, or single bilayer phosphatidylcholine vesicles, even though rate constants for benzo(a)pyrene transfer from these donor vehicles differed by 10-fold. When phosphatidylcholine vesicles containing benzo(a)pyrene and a nontransferable fluorescence quencher were mixed with cells in a stopped-flow system, two kinetic components were distinguished: a fast component with a rate constant corresponding to that measured for transfer of benzo(a)pyrene out of vesicles, followed by a much slower component, with a time course approximating that measured for cellular accumulation of benzo(a)pyrene by other techniques. Rate constants for desorption of a series of PAH which contained different number of aromatic rings from phosphatidylcholine vesicles differed over a 70-fold range. First-order rate constants for cell uptake of benzo(a)pyrene and five other PAH of different molecular sizes had the same 70-fold range of values, but were 2 orders of magnitude smaller than their respective rate constants for desorption from single bilayer vesicles. In addition, activation energies for cell uptake were essentially identical to the respective activation energies for desorption of PAH from phosphatidylcholine vesicles, confirming the mechanistic similarity of the two processes.     

Bacterial oxidation of the polycyclic aromatic hydrocarbons acenaphthene and acenaphthylene.

A Beijerinckia sp. and a mutant strain, Beijerinckia sp. strain B8/36, were shown to cooxidize the polycyclic aromatic hydrocarbons acenaphthene and acenaphthylene. Both organisms oxidized acenaphthene to the same spectrum of metabolites, which included 1-acenaphthenol, 1-acenaphthenone, 1,2-acenaphthenediol, acenaphthenequinone, and a compound that was tentatively identified as 1,2-dihydroxyacenaphthylene. In contrast, acenaphthylene was oxidized to acenaphthenequinone and the compound tentatively identified as 1,2-dihydroxyacenaphthylene by the wild-type strain of Beijerinckia. Both of these products were also formed when the organism was incubated with synthetic cis-1,2-acenaphthenediol. A metabolite identified as cis-1,2-acenaphthenediol was formed from acenaphthylene by the mutant Beijerinckia sp. strain B8/36. Cell extracts prepared from the wild-type Beijerinckia strain contain a constitutive pyridine nucleotide-dependent dehydrogenase which can oxidize 1-acenaphthenol and 9-fluorenol. The results indicate that although acenaphthene and acenaphthylene are both oxidized to acenaphthenequinone, the pathways leading to the formation of this end product are different.     

Characteristics of DNA during binding with polycyclic aromatic hydrocarbons in vitro]

Polycyclic aromatic hydrocarbons (PAH) were covalently bound to DNA by means of various activating systems. The following systems were used: the microsomal fraction of the rat liver, the system with I2, the system with ascorbic acid and FeSO4. Breaks in DNA due to the activating systems action appeared in all of these systems. Plateau of the PAH binding system curve in the microsomal system cannot be attributed either to the fall of the PAH metabolism rate to zero, or to the PAH binding sites in DNA. This plateau is the result of equalization of the rates of the two contrary-directed processes: the binding of metabolites and their removal due to DNA degradation. Because of the breaks in DNA caused by the activating systems, the authors failed to discover the changes in sedimentation data of DNA due to the covalently bound PAH.     

Non-enzymic activation of polycyclic aromatic hydrocarbons as mutagens.

Samples of 22 polycyclic aromatic hydrocarbons and related derivatives were subjected to ⁶⁰Co gamma radiation in air, and the irradiated samples were tested for mutagenicity with the Salmonella typhimurium strains TA 98, TA 1535, TA 1537, and TA 1538. Testing was conducted with the bacterial strains alone, thus not fortified with liver-microsomal enzymes or other metabolizing systems. Marked mutagen responses were obtained for several irradiated samples with the TA 98, TA 1537, and TA 1538 strains but not with the TA 1535 strain. Irradiated samples of benzo[a]anthracene, benzanthrone, benozo[g,h,i]perylene, benzo[a]pyrene, chrysene, fluorene, 9-methylanthracene, 1-methylphenanthrene, 2-methylphenanthrene, and pyrene gave positive mutagenic tests and dose-responses, whereas unirradiated control samples of these were inactive. Acenaphthene, phenanthrene, and phenanthrenequinone exhibited toxicity which interfered with interpretation of mutagenicity testing. Samples of 2-methylanthracene and tetracene were mutagenic with or without irradiation. Alizarin, anthracene, anthraquinone, anthrone, dobenzo[a,h]anthracene, picene, and triphenylene negative results. Samples of benzo[a]pyrene adsorbed on silica gel irradiated in air by ⁶⁰Co gamma radiation or by 254 nm ultraviolet light and samples adsorbed on filter paper irradiated by visible light yielded preparations mutagenic towards the TA 98, TA 1537, and TA 1538 strains. These results suggest that parent polycyclic aromatic hydrocarbons not themselves mutagenic towards S. typhimurium may be oxidized in air by radiation-induced processes to products whose mutagenicity resembles that of liver-microsomal metabolites of the parent polycyclic aromatic hydrocarbon.     

Heavy metal ions affecting the removal of polycyclic aromatic hydrocarbons by fungi with heavy-metal resistance

The co-occurrence of polycyclic aromatic hydrocarbons (PAHs) and heavy metals (HMs) is very common in contaminated environments. It is of paramount importance and great challenge to exploit a bioremediation to remove PAHs in these environments with combined pollution. We approached this question by probing the influence of HMs coexisting with PAHs on the removal of PAHs by Acremonium sp. P0997 possessing metal resistance. A removal capability for naphthalene, fluorene, phenanthrene, anthracene, and fluoranthenepresentalone (98.6, 99.3, 89.9, 60.4, and 70 %, respectively) and in a mixture (96.9, 71.8, 67.0, 85.0, and 87.9 %, respectively) was achieved in mineral culture inoculated with Acremonium sp. P0997, and this strain also displayed high resistance to the individual HMs (Mn²⁺, Fe²⁺, Zn²⁺, Cu²⁺, Al³⁺, and Pb²⁺). The removal of individual PAHs existing in a mixture was differently affected by the separately tested HMs. Cu²⁺enhanced the partition process of anthracene to dead or alive mycelia and the contribution of the biosorption by this strain but imposed a little negative influence on the contribution of biodegradation to the total removal of anthracene individually in a culture. However, Mn²⁺ had an inhibitory effect on the partition process of anthracene to dead or alive mycelia and decreased the contributions of both biosorption and biodegradation to the total anthracene removal. This work showcased the value of fungi in bioremediation for the environments with combined pollution, and the findings have major implications for the bioremediation of organic pollutants in metal-organic mixed contaminated sites.     

The micronucleus assay in exfoliated buccal cells: application to occupational exposure to polycyclic aromatic hydrocarbons.

Many polycyclic aromatic hydrocarbons (PAHs) have been identified as cancer-inducing chemicals for animals and/or humans. Also, there is sufficient evidence that exposures in the occupational settings are carcinogenic or probably carcinogenic to human. Engine exhaust and used engine oils are major PAH sources in engine repair workshops and traffic. Analysis of micronucleus (MN) in exfoliated buccal cells is a sensitive method for monitoring genetic damage in human populations. In our study, we used three different occupational groups (Group 1; engine repair workers, Group 2; taxi drivers, Group 3; traffic police) and two controls (Control I for Group 1 and Control II for Group 2 and Group 3) for the exposed groups. We analysed MN frequencies in exfoliated buccal cells and compared the exposed groups (Group 1; n=34, Group 2; n=17, Group 3; n=15) and subjects not occupationally exposed to PAH (Control I; n=28, Control II; n=20). The mean (+/-S.D.) MN (%) frequencies in exfoliated buccal cells from Group 1 and Control I were 0.07+/-0.05 and 0. 05+/-0.04, respectively (p>0.05; Table 2). The mean (+/-S.D.) MN (%) frequencies in exfoliated buccal cells from Group 2, 3 and Control II were 0.12+/-0.05, 0.10+/-0.05 and 0.03+/-0.03, respectively (p<0. 0001, p<0.05; Table 2) Smokers and nonsmokers do not differ with respect to the incidence of MN in all groups.     

Fungal metabolism and detoxification of polycyclic aromatic hydrocarbons

The mutagenic activity of ethyl acetate extracts of culture medium from Cunninghamella elegans incubated 72 h with various polycyclic aromatic hydrocarbons (PAHs) was evaluated in the Salmonella typhimurium reversion assay. All of the PAH extracts were assayed in tester strains TA98 and TA100 both with and without metabolic activation using a liver fraction from Aroclor 1254-treated rats. None of the extracts from fungal incubations with the mutagenic PAHs, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene and benz[a]anthracene, as well as the non-mutagenic PAHs, naphthalene, phenanthrene and anthracene, displayed any appreciable mutagenic activity. In addition, time course experiments indicated that the rate of decrease in mutagenic activity in the extracts from cultures incubated with benzo[a]pyrene or 7,12-dimethylbenz[a]anthracene was coincident with the rate of increase in total metabolism. The results demonstrated the ability of the fungus C. elegans to detoxify known carcinogens and mutagens and suggests that this organism may play an important role in the metabolism and inactivation of PAHs in the environment.Abbreviations hplc high performance liquid chromatography - tlc thin layer chromatography - PAH polycyclic aromatic hydrocarbon     

Effects of co-occurring aromatic hydrocarbons on degradation of individual polycyclic aromatic hydrocarbons in marine sediment slurries.

Rates of polycyclic aromatic hydrocarbon (PAH) degradation and mineralization were influenced by preexposure to alternate PAHs and a monoaromatic hydrocarbon at relatively high (100 ppm) concentrations in organic-rich aerobic marine sediments. Prior exposure to three PAHs and benzene resulted in enhanced [14C]naphthalene mineralization, while [14C]anthracene mineralization was stimulated only by benzene and anthracene preexposure. Preexposure of sediment slurries to phenanthrene stimulated the initial degradation of anthracene. Prior exposure to naphthalene stimulated the initial degradation of phenanthrene but had no effect on either the initial degradation or mineralization of anthracene. For those compounds which stimulated [14C]anthracene or [14C]naphthalene mineralization, longer preexposures (2 weeks) to alternative aromatic hydrocarbons resulted in an even greater stimulation response. Enrichment with individual PAHs followed by subsequent incubation with one or two PAHs showed no alteration in degradation patterns due to the simultaneous presence of PAHs. The evidence suggests that exposure of marine sediments to a particular PAH or benzene results in the enhanced ability of these sediments to subsequently degrade that PAH as well as certain other PAHs. The enhanced degradation of a particular PAH after sediments have been exposed to it may result from the selection and proliferation of specific microbial populations capable of degrading it. The enhanced degradation of other PAHs after exposure to a single PAH suggests that the populations selected have either broad specificity for PAHs, common pathways of PAH degradation, or both.     

Biomarkers of carcinogenesis in humans exposed to polycyclic aromatic hydrocarbons

The frequencies of micronuclei (MN) in cytokinesis-blocked lymphocytes of 91 steel foundry workers were analysed. On the basis of ambient PAH levels at the work stands and 1-hydroxypyrene concentrations in the urine, the coke-oven workers were the most exposed as compared to the rollers reference group. The difference in results for the two groups studied was not statistically significant, although MN were slightly higher for coke-oven workers. The frequency of MN did not increase with exposure: after some increase in 1-10 years, a decreasing tendency was observed.     

污染土壤中多环芳烃生物降解的调控研究

选用温度、湿度、表面活性剂ＴＷ８０和ＣＮＰ比４个因素为调控因子,采用正交法进行周期为１５０天的实验研究．结果表明,３０天后,土壤中ＰＡＨｓ的降解率可达４４．５～７４．６％,６０天后,达７０．４～９３．７％,降解率的不同与调控条件显著相关．在此期间,降解最佳条件为４０℃,湿度２５％,ＣＮＰ比为１２０１０１,ＴＷ８０分别为２００～５００ｍｇ·ｋｇ－１．实验结束时,土壤中ＰＡＨｓ的降解率达９１．２～９９．８％．降解的最佳条件是４０℃,湿度１５％．经Ｒ值判别表明,不同时期各因子对ＰＡＨｓ降解影响有所不同．温度对ＰＡＨｓ降解影响较大,表面活性剂对土壤中ＰＡＨｓ的生物降解有调控作用．     

Metabolism of polycyclic aromatic hydrocarbons in cultured human leukocytes under genetic control

Summary Metabolism of the polycyclic hydrocarbon benzo(a)pyrene(BP) to watersoluble products was measured in cultured human leukocytes from 35 healthy volunteers. The classification into 3 different groups with respect to degree of BP metabolism fits that obtained in a previous population and family study, where only the ratio of aryl hydrocarbon hydroxylase (AHH) activity of 3-methylcholanthrene (3MC) treated cultures to the activity of control cultures was measured. These data support previous observations that metabolism of polycyclic hydrocarbons is under genetic control.

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Zusammenfassung In der vorliegenden Arbeit wurde der Metabolismus des carcinogenen Kohlen wasserstoffes Benzpyren zu wasserlöslichen Produkten in Leukocytenkulturen von 35 gesunden Individuen untersucht. Hinsichtlich der Geschwindigkeit im Benzpyrenmetabolismus können 3 Gruppen unterschieden werden. Diese Einteilung ist in völliger Übereinstimmung mit der einer Populations-und Familienstudie, wo das Verhältnis der Aryl-Hydrocarbon-Hydroxylase-Aktivität von 3-Methylcholanthren-behandelten Leukocytenkulturen zu der von Kontrollkulturen bestimmt wurde. Diese Ergebnisse stützen die Beobachtung, daß der Metabolismus von carcinogenen Kohlenwasserstoffen unter genetischer Kontrolle ist.

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This work was supported in part by NIH Research Grant GM 15597 and Grant Ke 217/1 Deutsche Forschungsgemeinschaft (DFG).