Induction of Cytochrome P4501A1 in Haemopoietic Stem Cells by Hydroxylated Metabolites of Benzene

Toxic. in Vim Vol. 9, No. 4. pp. 453457, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0887-2333/95 $99.50+ 0.00

Induction of Cytochrome P4501Al in Haemopoietic Stem Cells by Hydroxylated Metabolites of Benzene R. HENSCHLER*$ and H. R. GLATTT *Department of Hematology, Freiburg University Medical Center, Hugstetter Strasse 55, D-79106 Freiburg and tDepartment of Toxicology, Deutsches Institut fur Ernihrungsforschung, D-14458 Potsdam-Rehbriicke,

Germany

Ahatract-The ability of various metabolites of benzene to regulate the expression of cytochrome P-450 (CYP)lAl mRNA in human haemopoietic cells was investigated. CYPlAl mRNA was quantified using a Northern blot technique and high stringency hybridization with a 32P-labelled cDNA probe. Benz[a]anthracene (BA, 1 or lop@, used as a positive control, induced CYPlAl mRNA in two out of three human leukaemic haemopoietic stem cell lines (positive: KG-l, U937; negative: HL-60), as well as in long-term bone marrow cultures established from healthy volunteers. In KG-I and U937 cells, CYPl Al mRNA induction was studied in the presence of the benzene metabolites, hydroquinone (HQ), p-benzoquinone (BQ), phenol (PHE) and catechol (CAT). HQ and BQ induced CYPlAl mRNA when added at concentrations of 10On~ or more; CAT was active at a concentration of 1 PM, whereas PHE had almost no effect, even at the highest concentration used (I FM). Maximum mRNA levels induced by 1 PM HQ were seen at 6 and 12 hr after addition of inducers, and induction was detectable for at least 48 hr. Little, if any, cellular toxicity was seen in clonogenic assays of KG-l cells at concentrations of maximum induction. In conclusion, CYPlAl mRNA induction was demonstrated in haemopoietic cells; inducers for CYPlAl were not only a polycyclic aromatic hydrocarbon (BA), but also, unexpectedly, hydroxylated metabolites of benzene.

Introduction

Cytochrome P-450 (CYP)lAl is a member of the P-450 superfamily of monooxygenase enzymes catalysing biotransformation of endogenous substrates as well as xenobiotic compounds including drugs, pollutants and carcinogens (Guengerich, 1990; Nebert et al., 1990). CYPlAl has been reported to be induced by structurally diverse polycyclic aromatic compounds including polycyclic aromatic hydrocarbons, halogenated dioxins and biphenyls, aromatic amines and flavonoids (Safe, 1986). A cellular mechanism for induction of CYPl Al has been identified (Whitlock, 1990). It involves binding of the inducer to the cytosolic Ah receptor, translocation of the receptor-inducer complex into the nucleus, binding of this activated complex to specific regulatory DNA sequences (xenobiotic responsive elements) and transcriptional activation of corresponding genes. The expression of CYPlAl mRNA in haemopoietic cells has not been investigated so far. Furtherbenzene more, the influence of hydroxylated iAuthor for correspondence. BQ = p-benzokbbreviations : BA = benz[a)anthracene; CAT = catechol; CYPlAl = cytochrome quinone; P45OlAl; FCS = foetal calf serum; HQ= hydroquinone; PHE = phenol.

metabolites or quinones, known to accumulate specifically in bone marrow after benzene exposure (Kalf, 1987), on CYPlAl expression has not been addressed. We report here on the induction of CYPlAl RNA in haemopoietic cells of humans. Materials and Methods Cells

Cell lines KG-l, U937 and HL-60 were purchased from the American Type Tissue Culture Collection. Human primary bone marrow cells were obtained from healthy volunteers after informed consent. Ceil lines were cultured in RPM1 tissue culture medium (Gibco, Paisley, UK) in the presence of 20% heatinactivated (56°C 30 min) foetal calf serum (FCS) in a humidified atmosphere containing 5% CO*. For some experiments, cells were washed free from serum 1 day before exposure. Long-term bone marrow cultures were initiated from erythrocyte-depleted bone marrow aspirates (1 g sedimentation in 0.1% methylcellulose in RPM1 medium over 30min) and seeded at a density of 1.5 x lo6 mononuclear cells/ml in tissue culture flasks (25 cm2; Falcon) in Iscove’s modified Dulbecco’s medium (Gibco), supplemented with 10% preselected FCS (Gibco), 10% preselected horse serum (Sigma, Munich, Germany) and 5 x lo-’ M hydrocortisone (Sigma). FCS and horse 453

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R. Henschler and H. R. Glatt

serum were preselected for optimum capability to support bone marrow stroma formation in culture and colony formation from supernatants of human long-term bone marrow cultures. The cultures were incubated at 33°C and fed weekly by half medium changes until confluence of the stroma was reached. Cultures were used at an age of 4 wk.

Results Basal and induced expression and induction oj CYPIAI mRNA in haemopoietic cells

After exposure, cells were washed in the PBS. The ensuing pellet was lysed with 4M guanidinium isothiocyanate (Sigma) and RNA was extracted by the method of Chomczynski and Sacchi (1987). Samples were fractionated by electrophoresis through a 1% agarose gel in 0.02 M N-morpholino-propanesulfonic acid (Serva, Heidelberg, Germany), pH 7.0, and 0.66 M formaldehyde, transferred to nylon membranes (Schleicher and Schuell, Dassel, Germany) and hybridized to a minimum of lo6 cpm 32P-labelled cDNA probes/ml. A total of 50 ng cDNA probes was labelled with 32P dCTP (3000Ci/mmol, Amersham, Braunschweig, Germany) using the hexanucleotide primer technique (Feinberg and Vogelstein, 1983). The filters were washed to a final stringency of 0.1 x SSC (I x SSC is 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0) at 65°C and exposed to Kodak X-omat films with intensifying screens for l-3 days. The cDNA probes used were the 1.2 kb EcoRl-EcoRl fragment of human P4501Al (obtained from Dr D. W. Nebert, Cincinnati, OH, USA), the 0.8 kb BamHl-Pst 1 fragment of chicken cr-actin in pBR 322 (Schwartz et al., 1980) and the 1.2 kb Pst I-Pst 1 fragment of rat GAPDH in pUC 18 (Tso et al., 1985).

In most control experiments with KG-l and U937 cells cultivated in medium containing serum, the predicted 2.5 kb transcript could barely be detected in Northern blots (not shown). The same observation was made in long-term bone marrow cultures from three normal healthy individuals. However, a clear signal was sporadically detected, and the presence of this signal depended on the batch of serum used. After serum depletion for 16-24 hr, only minimal basal CYPI Al mRNA levels were seen in all experiments. Therefore, some experiments were conducted after overnight serum depletion. The exposure to IO pM BA for 6 hr in the presence of serum resulted in substantial induction of CYPlAl mRNA in KG-l myeloblasts (not shown) and U937 myelomonoblastic cells (Plate la). Neither basal expression nor induction of CYPlAl mRNA was detectable in the HL-60 promyelocytic precursor cell line (Plate la). In long-term cultures derived from primary bone marrow cells, BA also induced increases in CYPIAI mRNA (Plate la). The exposure of KG-I or U937 cultures to HQ induced significant elevations of CYPlAl mRNA (Plate la). In KG-I cells other metabolites of benzene were also tested. The exposure to HQ, BQ or CAT led to induction of CYPlAl mRNA, whereas the exposure to PHE only resulted in barely detectable elevations at high concentrations (Plate lb). The response after addition of HQ and BQ became effective at concentrations of 100 nM, whereas CAT induced CYPlAl transcripts only at the highest concentration used (1 PM). When cultures were serum depleted, the concentration yielding the highest induction was decreased by a factor of 10 compared with the experiments in the presence of serum. Induction decreased at doses above 10~~ in all cultures, and was no longer detectable at concentrations of 100~~ (not shown). The maximum response of CYPl Al mRNA induction in KG-l cells was observed 6-12 hr after exposure. Declining mRNA levels were measured 24 and 48 hr after exposure (not shown).

Clonogenic assays

Cytotoxicity during exposure to the chemicals tested

KG-l cells were exposed to the benzene metabolites for 6 or 24 hr in RPMI/FCS in tissue culture flasks and then plated after several washings at 1 x 103/well in 0.3% agarose (Difco Laboratories, Detroit, MI, USA) in RPMI/20% FCS and 10% conditioned medium from the 5637 cell line (a gift from Dr R. Pettengeli, Paterson Institute for Cancer Research, Manchester, UK) in 35-mm petri dishes. Cultures were incubated at 37°C in a humidified atmosphere and colonies of more than 30 cells were counted through a light microscope on day 12 after seeding.

Cell viability, as determined by trypan blue exclusion, was not detectably affected in any of the reported experiments. As a more sensitive assay for possible cytotoxic effects in KG-l cells, soft agar clonogenic assays were performed in the presence of conditioned medium from the human bladder carcinoma cell line 5637 as a source of growth factors. Measurements performed after exposure times of 6 hr (Fig. la) and 24 hr (Fig. lb) revealed no cytotoxicity for some treatment regimens, and a mild cytotoxicity for others (reduction of the colony number by up to one-third).

Chemicals

Benz[a]anthracene (BA), hydroquinone (HQ), p-benzoquinone (BQ), catechol (CAT) and phenol (PHE) were purchased at analytical grade from Sigma. BA was dissolved in dimethyl sulfoxide (whose final concentration in the medium amounted to 0.1 %, v/v); the other substances were dissolved in phosphate buffered saline (PBS) immediately before addition to the cultures. RNA preparation and Northern blotting

(a) KG-1 I

I a

HL-60

1

0 ::z Ea

I

-

CYPlAl

-

GAPDH

(b) HQ

BQ

CAT

PHE

Plate I. Northern blot analysis of CYPlAl mRNA in haemopoietic cells. Cells (10’) were exposed to the indicated concentrations of BA, BQ, HQ, CAT or PHE for 6 hr. Total RNA was extracted and analysed for CYPlAl mRNA content. Actin or GAPDH transcript levels were determined as a control for comparable RNA loading. (a) induction of CYPlAl transcripts in serum-supplemented cultures of KG-l, HL-60, U-937 cell lines and long-term bone marrow cultures (LTBMC); (b) induction of CYPlAl in KG-I cells after 24 hr serum depletion.

455

Cytochrome P4501AI expression in blood stem cells

Thus, serum might adsorb substantial proportions of the metabolites in culture, or chemically alter them. The decrease and lack of observable induction at concentrations above 10 PM was probably related to cellular toxicity, as actin mRNA was also diminished at these concentrations and a loss of cell viability occurred. Up to now, CYPlAl has not been demonstrated to hydroxylate benzene or metabolites thereof. Rather, CYP2El has been implicated in benzene metabolism and toxification. A possible role for CYPlAl in benzene metabolism remains to be investigated. Also, it remains to be shown whether this induction is involved in the aplasiogenic and leukaemogenic effects of benzene.

(a)

I

451

(b)

REFERENCES

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m

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CAT

PIE

Fig. 1. Clonogenic assays of KG-l cells. KG-I cells (5000) were plated in methylcellulose after exposure in liquid culture to 1 PM BQ, HQ, CAT or PHE for (a) 6 hr and (b) 24 hr. Colonies were counted on day 12. Values are means f SD of three incubations. Asterisks indicate significant differences from control (*P i 0.05; **P < 0.01; one-sided Student’s r-test). Discussion

We have demonstrated induction of CYPIAI mRNA in primitive haemopoietic cells. Surprisingly, not only BA, a classical inducer of CYPl Al, but also hydroxylated and quinone metabolites of benzene showed this effect. The ability of benzene metabolites to regulate expression of CYPl Al is unexpected, since they are small monocyclic molecules, whereas the known inducers are polycyclic aromatic compounds. Interestingly, however, Pickett and coworkers found that BQ is capable of activating both an antioxidant responsive element (ARE) and a xenobiotic responsive element (XRE) in the 5’ regulatory region of the glutathione S-transferase Ya subunit gene (Rushmore and Pickett, 1994). It remains to be investigated whether the same or a different mechanism is involved in the induction of CYPlAl by benzene metabolites. Induction of CYPlAl occurred at lower concentrations in the absence of serum than in its presence.

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