Mutation Research, 281 (1992) 11-16

11

© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00

MUTLET 00603

Detection of benzo[a]pyrene-diol-epoxide-DNA adducts in white blood cells of psoriatic patients treated with coal tar M. Paleologo 1, F.J. van Schooten 2, S. Pavanello ~, E. Kriek 2, M. Zordan ~, E. Clonfero 3, C. Bezze 4 and A.G. Levis 1 I Department of Biology, "~Institute of Occupational Health and 4 Dermatology Clinic, Unicersity of Padua, Padua (Italy) and 2 Dit,ision of Chemical Carcinogenesis, Netherlands Cancer Institute, Amsterdam (The Netherlands)

(Received 23 July 1991) (Revision received 2 September 1991) (Accepted 12 September 1991)

Keywords: DNA adducts; Benzo[a]pyrene-diol-epoxide;Coal tar; Enzyme-linked immunosorbent assay; Psoriasis

Summary An enzyme-linked immunosorbent assay (ELISA) was used to detect B P D E - D N A adducts in white blood cells of 23 psoriatic patients undergoing clinical coal tar therapy. Ten of these patients were reanalyzed 2 - 5 months after the end of the coal tar treatments. The results show that the mean adduct level during the treatment period was 0.26_+0.16 fmole B P D E / p , g D N A (7.7 + 4 . 9 a d d u c t s / 1 0 8 nucleotides), while 2-5 months later the mean adduct level had decreased significantly ( P < 0.005) to 0.11 _+ 0.08 fmole B P D E / / ~ g D N A (3.3_+ 2.4 adducts/10 8 nucleotides). No relationship could be ascertained between the level of exposure and the amount of B P D E - D N A adducts. In addition, no difference in the level of D N A adducts was found between smoking and non-smoking patients.

Psoriasis is a common epidermal disease affecting about 3% of the world population. Subjects suffering from the more severe forms of this

Correspondence: Dr. S. Pavanello, Department of Biology, University of Padua, Via Trieste 75, 1-35121 Padua (Italy). Abbrel'iations: BP, benzo[a]pyrene; BPDE, (+)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene;

BPDE-DNA, DNA modified by the reaction with BPDE, forming the major adduct BPDE-dG; BSA, bovine serum albumin; CT, coal tar; ELISA, enzyme-linkedimmunosorbent assay; 4-MUP, 4-methylumbelliferyl phosphate; PAIl, polycyclic aromatic hydrocarbons; TO, ointment base containing 4% coal tar; TP, paste base containing 4% coal tar; WBC, white blood cells.

affection are often treated with coal tar-based ointments (Bridges et al., 1981). Coal tars (CT) were recently evaluated by I A R C as having sufficient evidence of carcinogenicity in humans (IARC, 1987). CT, including those employed for therapeutic purposes, generally have a high content of polycyclic aromatic hydrocarbons (PAH) and in particular of benzo[a]pyrene (BaP) (Lijinsky et al., 1963). Percutaneous absorption of CT results in the systemic distribution of PAH, as indicated by the detection of cytogenetic damage in the lymphocytes of coal tar-treated psoriatic patients (Sarto et al., 1989) as well as by the excretion of high levels of mutagenic compounds in their urine following therapy (Clonfero et al., 1989). Many human tissues are able to enzymati-

12 cally c o n v e r t

BaP

to m u t a g e n i c

(electrophylic)

b e n t a s s a y ( E L I S A ) in t h e p e r i p h e r a l W B C o f a

m e t a b o l i t e s , l e a d i n g to the f o r m a t i o n of covalent

g r o u p o f 23 m a l e p s o r i a t i c p a t i e n t s d u r i n g a n d

adducts with DNA, the biologically more relevant

2-5 months after treatment coal t a r - b a s e d p r e p a r a t i o n s .

o f w h i c h is t h o u g h t t o b e t h e a d d u c t f o r m e d b y the

interaction

of

with therapeutical

benzo[a]pyrene-diol-epoxide

(BPDE) with DNA. Human

m o n o c y t e s a n d lym-

Materials and methods

p h o c y t e s a r e also a b l e t o m e t a b o l i c a l l y a c t i v a t e B a P in v i t r o ( R f i d i g e r e t al., 1985; P a v a n e l l o e t

Chemicals

al., 19891. Several authors have reported the presence of

obtained

detectable amounts

heim,

of BPDE-DNA

a d d u c t s in

4-Methylumbelliferyl phosphate from

Boehringer

Germany).

(4-MUP) was

Mannheim

(Mann-

(+)-trans-7,8-Dihydroxy-anti-

t h e w h i t e b l o o d cells ( W B C ) o f w o r k e r s e x p o s e d

9 , 1 0 - e p o x y - 7 , 8 , 9 , 1 0 - t e t r a h y d r o b e n z o [ a ] p y r e n e (B-

t o P A H ( W o g a n , 1989; V a n S c h o o t e n e t al., 1990).

PDE) was obtained from the Midwest Research

In t h e p r e s e n t s t u d y B P D E - D N A

a d d u c t levels

w e r e m e a s u r e d by a n e n z y m e - l i n k e d i m m u n o s o r -

Institute

(Kansas

City,

MO,

U.S.A.).

Bovine

s e r u m a l b u m i n ( B S A ) , g o a t a n t i - r a b b i t I g G alka-

TABLE I AGE, SMOKING HABITS, TREATMENT PROTOCOL FOLLOWED, AND LEVELS OF BPDE-DNA ADDUCTS OF 23 PSORIATIC PATIENTS FOLLOWING THERAPY WITH COAL TAR Patient

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Age (years)

51 25 37 56 58 47 NA NA 23 NA 24 58 60 16 48 60 34 45 58 31 52 42 40

Smoking habits "

S S S NS NS NS NA NA S NA NS NS NS NS S S S S NS NS S S S

Days of treatment

8 7 6 6 13 8 6 13 7 7 10 8 6 3 17 9 5 11 6 5 10 10 10

Type of treatment b

TP TP CT, TP TP TP CT, CT, TP CT TP TP TP TP CT, TP TP CT, TP TP TO TO TO

TP

TP TP

TP

TP

% Body surface with lesions

75 80 80 65 35 80 40 20 35 60 20 30 70 75 20 70 70 30 60 60 80 80 80

~ S, smoker; NS, non-smoker. b CT, pure coal tar; TP. coal tar-based paste; TO, coal tar-based ointment. 1 fmol/mg DNA = 30 adducts/10 s nucleotides. d During or immediately after coal tar therapy. ~" 2-5 months after the end of coal tar therapy. NA, data not available.

Mean level of adducts ( + SD) (fmole/p.g DNA c) During d

Months later c

0.28 (0.08) 0.15 (0.05) 0.25 (0.10) 0.21 (0.08) 0.11 (0.09) 0.36 (0.23) (I.14 (0.03) 0.26 (0.191 0.50 (0.22) 0.40 (0.11 ) 0.24 (0.13) 0.28 (0.09) 0.51 (0.26) 0.28 (0.17) 0.24 (0.10) 0.10 (0.10) 0.13(0.11) 0.16 (0.16) 0.07 (0.07) (/.15 (0.101 0.15 (0.09) 0.12 (0.07) 0.10 (0.04)

0.06 (0.(13) 0.15 (0.04) 0.05 (0.03) 0.03 (0.01) 0.15 (0.16) 0.24 (0.12) 0.22 (0.04) 0.10 (0.08) 0.02 (0.01) 0.07 (0.05) -

13

Modification of DNA with BPDE and 3H-BPDE

line phosphatase conjugate, proteinase K and RNAse were purchased from Sigma (St. Louis, MO, U.S.A.). 3H-BP (23.8 C i / m m o l e ) and 3HB P D E (23.7 C i / m m o l e ) were purchased from A m e r s h a m International (Amersham, U.K.).

Highly modified B P D E - D N A (42 p m o l e / / z g DNA) and low-modified 3 H - B P D E - D N A were prepared as described by Van Schooten et al. (1987).

Subjects analyzed

Cell and DNA isolation

Twenty-three male psoriatic patients, presenting cutaneous lesions involving 20-80% of the body surface, excluding the genital area and hands, were analyzed. The patients were hospitalized in the Dermatology Clinic of the University of Padua (Italy). For each patient data regarding age, smoking habits, occupational or consistent environmental exposure to PAH, medical history, domiciliary treatments for psoriasis and drugs taken during the coal tar therapy were collected, the most relevant of which are reported in Table 1. For patients 7, 8 and 10 the above information was not available. During hospitalization, 6 of the patients were treated with crude coal tar either alone or in association with a coal tar-based paste (TP, containing 4% coal tar); 14 of the patients were treated with TP alone, while the remaining 3 subjects received treatment with a 2% coal tar ointment (TO). Finally, 10 of the patients were reanalyzed 2 - 5 months after the end of the clinical coal tar treatments.

Samples of peripheral blood (20-40 ml) were collected by venepuncture in heparinized plastic syringes. The purity of D N A isolated from cell lysates by cycles of p h e n o l / c h l o r o f o r m extraction was checked and quantified by U V spectroscopy (the absorbance ratios at 260:280 nm were always greater than 1.75).

Detection of BPDE-DNA adducts ELISA assays were performed as described by Van Schooten et al. (1987) using a rabbit anti B P D E - D N A polyclonal antiserum (F29). H u m a n D N A samples were tested in 2 - 6 separate experiments (depending on the amount of D N A available), in addition each sample was assayed in triplicate or quadruplicate within each experiment. Different concentrations of 3 H - B P D E D N A standard were assayed in each experiment in quadruplicate. All assays were performed blindly with coded samples. TRANSFORMED DATA

ORIGINAL DATA

lo0

i00

Q

9o

-

90.

80

80.

70

70.

60

60.

50

50.

40