M.W.A.C. Hukkelhoven

A sensitive fluorometric assay for epoxide hydratase

It is now firmly established that a great number of xenobiotics, e.g., drugs, environmental pollutants and food additives can be metabolized by microsomal cytochrome P450-dependent monooxygenases to highly reactive epoxide intermediates [ 1,2]. Epoxide hydratase (EC 3.3.2.3.) has a dual role in the further metabolism of these epoxides. On the one hand, hydration of arene oxides to the chemically less reactive truns-dihydrodiols can be considered as a detoxification pathway, while on the other hand certain trans-dihydrodiols of PAH can be further oxidized by the monooxygenases to highly mutagenic and carcinogenic diol-epoxides [3,4]. Since inter-individual differences in carcinogen metabolism can result in different susceptibility to obtain carcinogen-induced neoplasm, it is of interest to detect genetic differences in activity of the various carcinogen metabolizing enzymes [5,6]. Human hair follicles have been suggested as a convenient biopsy-tissue for these investigations [7,8]. Here, we present a rapid and very sensitive fluorometric assay for EH, using 4,5-dihydro-epoxyBP as substrate, which is especially useful for hair follicles and for other small tissue samples with relatively low EH activities.

Determination of phenolic benzo(a)pyrene metabolites formed by human hair follicles

Abstract A modified fluoreometric procedure for the determination of basal arly hydrocarbon hydroxylase activity in human hair follicles is described. The method is also applicable for measurement of induction of the enzyme in primary cultures of keratinocytes originating from hair follicles. For good sensitivity of the assay the use of small incubation and extraction volumes, ultrapure chemicals, and adaptations in the measurement of the fluorescent signal are required. Moreover a sensitive micromethod for measuring DNA as a reference variable has to be employed. The sensitivity of the assay permits the measurement of aryl hydrocarbon hydroxylase in 20 hair follicles or in the outgrowth of 6 cultured hair follicles with good reproducibility. The method gives the opportunity to identify individuals with genetically controlled differences in the metabolism of polycyclic aromatic hydrocarbons, using an easily obtainable biopsy tissue of epithelial origin.

Human hair follicles and cultured hair follicle keratinocytes as indicators for individual differences in carcinogen metabolism.

Benzo(a)pyrene (BP)-metabolism in freshly isolated human hair follicles, cultured hair follicle keratinocytes and cells cultured from human bronchial epithelium was analysed by high performance liquid chromatography. All three types of tissues resulted in quantitatively comparable amounts of the most important organic solvent-soluble metabolites: 9,10-dihydrodiol-BP, 7,8-dihydrodiol-BP, quinones, and phenols. Besides these metabolites two early eluting compounds were detected: one possibly is BP-3-yl-hydrogen sulfate, the other probably consists of one or more tetrols. Water-soluble metabolites were quantitatively unimportant in both types of cultured cells and appeared to be primarily glucuronide and sulfate conjugates with the monohydroxides and the 7,8-dihydrodiol of BP. This metabolic pattern is compared to that of monocytes and lymphocytes which have been used frequently in population studies and with data from other types of human epithelial cells. It is concluded that human hair follicles and cultured keratinocytes from these organs are useful for detection of individual differences in carcinogen metabolism.

Gene expression of transformed lens cells.

In addition to our SV40-transformed hamster cell line (Bloemendal et al. 1980), two otherlenticular cell lines have been established. One was obtained by transformation of hamster lens cells with the carcinogen 3-methylcholanthrene, the other one by transformation of calf lens cells with SV40. The transformed cells revealed alterations in growth pattern and morphology, but only minor changes in gene expression. However, crystallin synthesis ceased in all cases. We hypothesize that elongation of the lens cells in vitro reflects a transition into fibroblast-like cells.

Covalent binding of BP-metabolites to DNA of cultured human hair follicle keratinocytes

Primary cultures of human hair follicle keratinocytes were established by using a basement membrane-like growth substrate, the bovine eye lens capsule. A method was adapted for the isolation of 3H-benzo(a)pyrene (BP)-modified DNA from the cellular outgrowth of only one hair follicle (approximately 2×105 cells). In a routine procedure hair follicle keratinocytes were incubated with 0.5 μM 3H-BP for 24 h. The purified DNA was subjected to enzymic hydrolysis and the adducts were analyzed by Sephadex LH-20 column chromatography followed by HPLC. Only one major adduct, which represented 60–80% of the total radioactivity which can be confined to modified nucleosides in the LH-20 chromatograph, could be identified. This adduct co-chromatographed with the marker adducts resulting from the trans-addition of the N-2-amino group of guanine to the 10-position of (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-etrahydrobenzo(a)pyrene.Co-incubation with 7,8-benzoflavone (0.3 μM), an inhibitor of cytochrome P-448, and with 1,1,1-trichloropropene-2,3-oxide (0.2 μM), an inhibitor of epoxide hydrolase, resulted in a marked inhibitory effect (15% of the control binding) and a large increase (300% of the control value) in BP-DNA binding respectively. Induction of aryl hydrocarbon hydroxylase activity in the cultures with 5,6-benzoflavone (10 μM) or benz(a)anthracene (10 μM) caused a decrease (75 and 46% of the control value respectively) in BP-DNA binding. The ratio between “total” binding (as calculated from the specific activity of the isolated DNA) and “true” binding (represented by the BP-nucleoside adducts eluted from the HPLC column) appeared to be rather constant in cultures from four different individuals. Therefore, total binding may serve as a good representative of true binding. Using cultures from hair follicles of eight different persons, interindividual variation was 4-fold, with a mean binding of 2.4×109 molecules/μg DNA. Since it has been demonstrated that the formation of dihydrodiols of BP — among them the proximate carcinogen 7,8-dihydrodiol-BP — in freshly isolated hair follicles is largely genetically determined (Hukkelhoven et al. 1982), the interindividual differences in binding may possibly reflect individual variation in susceptibility to BP-induced neoplasia.

Metabolism of benzo(a)pyrene in bovine lens epithelium.

Bovine lens epithelium contains an enzyme system that metabolizes the carcinogen benzo(a)-pyrene. The major ethylacetate-soluble metabolites are 7,8-dihydro-7,8-dihydroxybenzo(a)-pyrene, 9,10-dihydro-9,10-dihydroxybenzo(a)pyrene and 3-hydroxybenzo(a)pyrene. Addition of 1,1,1-trichloropropene-2,3-oxide (TCPO), an inhibitor of expoxide hydratase, prevents the formation of the dihydrodiols. The overall metabolism can be inhibited by the addition of α-naphthoflavone. The metabolism of benzo(a)pyrene in cultured lens epithelial cells has also been studied. It is found that the activity of aryl hydrocarbon hydroxylase (AHH) is strongly decrease in culture. The decline in AHH activity can be reduced by the addition of an unphysiological concentration of nicotinamide.

Cytoskeletal Structures in Cultured Bovine Lens Cells and their Role in Lens Cell Elongation

Polygonally shaped epithelial lens cells show elongation upon subculturing. As a result cytoskeletal structures change in relative abundancy. A cytoskeletal constituent, occurring in the perinuclear p

Differences in benzo(a)pyrene metabolism between cultured human and murine bronchial cells after pre-treatment with benz(a)anthracene

Primary cultures of human and murine (strain C3Hz) bronchial epithelial cells were pretreated with benz(a)anthracene (BA) (10 microM). 16 h later the formation of phenolic as well as dihydrodiol metabolites of benzo(a)pyrene (BP) was measured. Whereas murine cultures showed enhanced metabolism towards both phenolic and dihydrodiol compounds, in the human cultures only phenolic BP-metabolites were increased. In view of their precursor role in the formation of biologically active diol-epoxides, formation of dihydrodiol-derivatives can be considered as a key factor in determining susceptibility to polycyclic aromatic hydrocarbon (PAH)-induced carcinogenesis. Therefore the observations of this study indicate that animal model systems for PAH carcinogenesis in man have to be selected on the basis of comparable metabolite patterns.

In vivo induction of aryl hydrocarbon hydroxylase in human scalp hair follicles by topical application of a commercial coal tar preparation

Five low-dose applications of a commercial coal tar preparation on a small scalp skin region resulted in an induction of aryl hydrocarbon hydroxylase (AHH) activity in freshly isolated human hair follicles. Large but reproducible interindividual differences in AHH-inducibility could be detected. The method offers the opportunity to measure AHH-inducibility, which has been correlated to the risk of developing chemical-induced cancer, in vivo in normal epithelium, a cell-type highly relevant for chemical carcinogenesis. Smoking habits did not have any effect on AHH-activity in freshly isolated hair follicles. Therefore the method potentially permits the identification of persons with high and low genetically determined AHH-inducibility.

Formation of phenolic and dihydrodiol metabolites of benzo(a)pyrene in freshly isolated human hair follicles occurs independently

Freshly isolated hair follicles of 20 adult non-smoking volunteers were assayed for formation of phenolic and dihydrodiol metabolites of benzo(a)pyrene (BP). In each of a total of 14 experiments two volunteers were assayed simultaneously, and the ratios of both phenolic and dihydrodiol metabolites of BP between the two individuals were determined. It was obvious that the mean interindividual variation in formation of phenolic metabolites was greater than the variation in formation of dihydrodiol metabolites. No correlation existed between the amount of both types of metabolites formed. These observations indicate that for detection of differences in carcinogen metabolism to assess individual susceptibility to the carcinogenic action of polycyclic aromatic hydrocarbons (PAH), measurements of phenolic and dihydrodiol metabolites of BP are not interchangeable.