Organ-specific and transplacental DNA damage and its repair in rats treated with 1,2-dibromo-3-chloropropane

ELSEVIER

Chemico-Biological Interactions 101 (1996) 33-48

Organ-specific and transplacental DNA damage and its repair in rats treated with 1,2-dibromo-3chloropropane

Rcccivcd 29 Scptcmhcr

1995: rcviscd 26 March

1996: acccptcd 2% March

1996

Abstract An in vivo genotoxicity formation

assay system based on alkaline elution has been used to study the

and removal of DNA

Cells/nuclei from different ing/homogenization

damage induced by I ,2-dibromo-3-chloropropanc

(DBCP).

tissues and organs of Wistar rats were prepared by a rapid minc-

technique.

organs of the same animal.

Thirty-six

samples of which

were then assayed in parallel

strand breaks plus alkali-labile

up to I I were from different

for DNA

sites = SSBs) with a semi-automated

damage (DNA

A single i.p. injection of DBCP gave dose- (5 and IO mg/kg) and time- (20 min-4 SSBs in kidney and liver DNA

from male rats. At 10 mg/kg DBCP.

single-

alkaline elution system. h) dependent

SSBs were formed in all

organs examined except the bone marrow and colon: however. an increased dose of 40 mg/kg produced SSBs also in the latter two organs. The relative susceptibilities DNA

damage were: kidney -

dular stomach > spleen -

duodenum

testis > bone marrow

previous data on tissue distribution a single i.p. dose of DBCP. hepatotoxic

> liver > lung -

brain -

in the liver and kidney after DBCP

plus alkali-labile

and organ necrosis in liver, kidney and testis of rats given similar levels of SSBs were detected in the livers

PII:

I .3-dibromo-3-chloropropanc:

Normalized

changes in SSBs were followed

exposure. In both organs SSBs peaked around 4 h post-

GSH.

glutathionc:

Area Abovc Curve: PB. phenobarbital:

SSBs. DNA

sites.

* Corresponding

0009-2797/96/$15.00

DBCP,

author. Tel.: 47 22 042426: fax: 47 22 041686.

0

1996 Elsevier Science Ireland Ltd. All rights rcwrvcd

SOOO9-2797(96)03709-X

glan-

When female rats were injected i.p. with 5. IO or 70 mg/kg (non-

doses) at day 20 of pregnancy.

Ahhwritrriorrs:

-

- colon. These relative levels correlate with

of the dam and the fetuses. In adult male rats. time-dependent

transferase: NAAC.

to DBCP-induced

urinary bladder

GST.

glutathionc-S-

single-strand breaks

34

G. Bnmborg et al. /Chemico-Biological

exposure, 50% had

been removed

ed to control levels. Pretreatment the maximum level of DNA

tion characteristics in various

organs

of rats with phenobarbital

prior to DBCP exposure reduced

damage as well as its persistence. In cultured primary hepatocytes (2-20

PM,

I h). 50% of the initial DNA

damage

- IO0 min. In conclusion. the experiments indicate that the distribu-

of DBCP

are of major importance

for DNA

of rats. The data are also in accordance

rather than P450. being the most important extrahepatic

33-48

by 12-24 h. whereas at day 2-3 SSB frequencies had return-

from male rats exposed in vitro to DBCP had been repaired within

Interactions 101 (19%)

tissues including

damage and its persistence

with glutathione&transferase,

pathway for metabolic activation

the fetal liver. It appears that alkaline

of DBCP

in rat

elution of cells/nuclei

prepared from exposed animals constitutes a sensitive, rapid and versatile technique to study organ- and cell-specific genotoxicity

in vivo.

Keywortls: Organ-specific;

Transplacental;

3-chloropropane;

elution

Alkaline

Genotoxicity:

DNA

strand breaks: I .2-Dibromo-

1. Introduction

The initial level and type of induced DNA modification. its rate of removal as well as the intragenomic localization, are considered to be essential features in the initiation of cancer [I]. DNA damage also plays an important role in cell death and organ toxicity [2,3], as has been suggested for several compounds including l,2-dibromo-3chloropropane (DBCP) [4]. DBCP was widely used as a soil fumigant against nematodes until it was reported to induce irreversible sterility amongst male production workers [5]. A developmental toxicity is indicated by an altered sex ratio in the offspring of DBCP-exposed males [6]. In the rat, which is the most sensitive animal species, single injections of DBCP have been shown to cause testicular atrophy and necrosis of the seminiferous epithelium [7,8]; necrosis has later been demonstrated in a range of other organs [9]. Intrauterine DBCP exposure may affect the development of seminiferous tubules in rats [IO]. DBCP is genotoxic in several in vitro and in vivo bacterial and mammalian test systems (reviewed by Dybing et al. [I I]) and is classified as a multi-organ animal carcinogen and a possible human carcinogen [12]. The compound is converted into DNA-reactive metabolite(s) probably via P450 and/or glutathione-S-transferases (GSTs) [ 1 I]; however, the relative roles of these pathways for DNA damage and necrosis in different organs are not known in detail. Information on the organotropic formation and removal of DBCP-induced DNA damage may contribute to a better understanding of the relation between such damage and the organ necrosis, cancer and reproduction toxicity associated with DBCP exposure. A number of different tests are available to assay in vivo for genotoxicity [ 131; however, most of these may be used with only a few cell types or organs from experimental animals. Based on the original alkaline elution assay of Kohn et al. [14] we have developed a semi-automated set-up with a high capacity for the analysis of DNA single-strand breaks and alkali-labile sites (SSBs) [ 151. When combined with rapid non-enzymatic techniques for the preparation of cells/nuclei, multiple organs from exposed animal(s) may be analyzed in parallel for DNA damage [l6-181. In

G. Bnmbog et al. /Chemico-Biological Interactions 101 (19%) 33-48

35

the present study we have used this approach to characterize DBCP-induced DNA damage in several organs, including the fetal liver. of Wistar rats after a single i.p. treatment. 2. Materials and methods

DBCP synthesized as described [I91 was a gift from Dr. S.D. Nelson. University of Washington, Seattle; its purity was above 98% as determined with gas chromatography. Media and chemicals were from the following sources: Collagenase (CLS II, I50 U/mg) from Worthington Biochemical Corp. (Freehold, NJ): Dulbecco’s modified Eagle’s medium without cysteine from the National Institute of Public Health, Oslo, Norway; Hoechst 33258 (2[2-(4-hydroxyphenyI)-6-benzimidazole-6-( lmethyl-4-piperazyl)-benzimidazole trihydrochloride)) from Calbiochem-Boehringer (La Jolla. CA); proteinase K from Merck (Darmstadt, Germany); trypsin and bovine serum albumin (V) from Sigma Chemical Company (St. Louis, MO); RPM1 1640 w/Hepes culture medium, Minimal essential Medium (MEM), fetal calf serum and antibiotics from Gibco BRL (Grand Island, NY). Other chemicals were commercial pa grade. 2.2. Animals Male and female Wistar rats (Mol:WIST, 200-300 g) from Mdlegaard Breeding and Research Center (Ejby, Denmark) were housed in plastic cages on hardwood bedding, and were given standard pelleted feed (RMI(E), Special Diet Services, Wilham, Essex, UK) and water ad libitum. Pregnant animals were obtained at day 12 and were treated with DBCP at day 20 (i.e. I day before normal delivery). 2.3. Clwmid

trwtmtwt

q/’ unimuls

untl organ procvssin~

Animals (males, or pregnant females) were given single intraperitoneal injections (0.5 ml/200 g) of DBCP dissolved in DMSO. Some animals were pretreated with i.p. injections of phenobarbital (PB, 75 mg/kg) dissolved in saline, for 3 consecutive days followed by the DBCP treatment on day 4. At specific times animals were anesthetized with diethylether, and up to I I different organs from each animal were removed and treated as follows [16]: the liver, kidney, testis, lung and spleen were minced briefly with scissors in ice-cold Merchant’s Medium with IO mM EDTA (M-EDTA; 0.14 M NaCI, 1.47 mM KH2P04, 2.7 mM KCI, 8.1 mM Na2HP04, IO mM Na2EDTA, pH 7.4), rinsed, and by means of a plastic plunger, forced through a stainless-steel screen (0.4 mm) fitted to a I5 mm i.d. stainless-steel cylindrical tube machined for this purpose. Thereafter the suspension was filtered through one layer of cotton gauze. The glandular (pyloric) part of the stomach was vigorously shaken in PBS, and the duodenum, colon ascendens and the urinary bladder were rinsed with M-EDTA; the layer of epithelial cells of these organs was scraped off in M-

36

G. Bnmborget aI. /Chemico-Btolo#calInteractions101 (19%) 33-48

EDTA with a glass microscope slide and then briefly homogenized in a glass homogenizer with a loose-fitting pestle. The femur was opened and bone marrow cells were rinsed out and homogenized. The brain was homogenized directly. When using pregnant females, liver samples of the dam plus the livers of 3-5 live fetuses were removed, pooled and processed as described above. Care was taken to keep samples ice-cold and under dim light at all stages during their preparation. All samples were then centrifuged (100 x g, 4 min), the supernatants discarded and the loose pellets were resuspended in M-EDTA. After filtration (0.25 mm nylon mesh), counting and-dilution, 2 ml (OS-4 x lo6 cells or nuclei) were loaded onto each filter of the alkaline elution system. The processing of 36 samples as described takes -2 h. 2.4. Chemical treatment of primary hepatocytes in vitro For each experiment hepatocytes were prepared from one male rat by a collagenase perfusion method [20] as previously described [21]. Cell viability at the start of an experiment was above 95% as determined by trypan blue exclusion. Hepatocytes (2 x 106/ml in MEM + 1% bovine serum albumin) were exposed to DBCP (O-20 PM) dissolved in DMSO at a final concentration of 0.25’%, in scintillation vials with shaking at 37°C. The treatment was terminated by cooling and washing, whereafter cells were resuspended in RPM1 1640 medium w/Hepes, supplemented with 20% fetal calf serum and antibiotics (= culture medium) at l-2 x 106/ml. Two ml of this suspension per 6-cm bacterial petri dish was incubated for repair in a CO2 incubator at 37°C. Samples were then cooled on ice and analyzed for DNA damage with alkaline elution. 2.5. Analysis of DNA single-strand breaks with alkaline elution DNA damage was assayed as SSBs with a semi-automated alkaline elution procedure [15] based on the method of Kohn et al. [l4]. Elution was with 0.02 M NazEDTA pH 12.50 f 0.03, for 16 h at 0.035 ml/min. The DNA contents of eluted fractions were assayed fluorimetrically in an automated setup, using the fluorochrome Hoechst 33258 and a Perkin Elmer LSSO (Norwalk, CT) luminescence spectrometer, as previously described [ 151. Elution profiles often deviate from straight (semi-logarithmic) lines, and an estimation of damage levels from such profiles is not always straightforward. Several methods have been proposed [22,23]. An alternative approach, which is routinely used in our laboratory, takes into account more of the information intrinsic in the elution profile. The calculation is based on the integrated area above the profile: ‘mm

Area =

IS r=O

In(R(t))dt I

where R(t) = DNA retained on filter at time t, and tmax= total elution period. By replacing the integral with a sum of discrete measurements, and by multiplying the

G. &unborg et al. I Chemico-BiologicalInteractions Ml (19%) 33-48

37

resulting expression with the constant 2/(1,&l, one obtains a normalized value (i.e. not dependent on the duration of elution), which we term the ‘Normalized Area Above Curve’ (NAAC):

NAAC =

II-l

1

[lnR,+lnRi + t]

c

n.&x

I

i=O

or, in a more convenient form for calculation: NAAC =

1

n Llax

,tI I

c I= I

(2lnRJ + InR, I

Here, n = total number of eluted samples, and R; = DNA fraction retained after eluted sample number i. This ‘area’ has the same dimension (h-l) as the first-order elution rate constant, and NAAC is identical to the latter constant when the semilogarithmic elution profile is straight-lined. This method represents a convenient way to quantify elution profiles. However, NAAC values must be used with care when comparing elution curves with greatly differing shapes [ 161. In the present report, NAAC values and error bars in the figures and Table 1 are means + S.E.M. of at least 3 parallel samples from different animals or in vitro incubations. Calibration of the alkaline elution setup is routinely carried out, using different mammalian cell types, with X-rays (260 kV maximum energy, 0.5 mm Cu filter). An averaged yield is NAAC = (23 f 2) x IO-‘h-’ per Gy (based on 2.7 x lO-‘(’ SSBs/Gy/Dalton [ 14]), or about 1000 SSBs/Gy/diploid cell. Converted into SSBs per Dalton: 1 NAAC (lo-‘h-l) = 11.4 SSBs/lO”Da. The sensitivity of this system to detect induced strand breaks depends on the background level in unexposed cells. In the present study, control values (NAAC, x lo-sh-‘) were between 2.06 f 2.81 (liver) and 15.56 f 7.69 (kidney), which means that approximately 150 and 1000 SSBs per cell should be detectable in the liver and kidney, respectively. 2.6. Statistical analysis NAAC values were tested for statistical significance using the Student’s l-test (unpaired, two-tailed). 3. Results Relatively low control SSB levels were recorded in organs from unexposed rats (Figs. 1 and 2). Attempts to prepare nuclei from the forestomach were unsuccessful (always very high break levels in controls; data not shown); with this exception all other tissues/organs tested gave nuclei with apparently largely intact DNA. Lowest control break levels were obtained with the liver and brain (23 f 32 and 24 f 31 SSBs per lO”Da, respectively), representing O-5 SSBs per average chromosome.

G. Brunborg

38

et al. / Chemico-Biological

Inferactions

33-48

101 f 19%)

1lVER

KIONEY

0.6 -

0.2 10 mg/kg

5 mg/kg

t

Elution Fig. I. Dose- and time-dependent

DNA

I

I

I

1

L

4

6

8

10

time

t

I

12 14

I

16

(hrs) Male rats were treated with DBCP

or 4 h post-exposure.

Liver and kidney nuclei were assayed

SSBs with alkaline elution. The numbers indicate NAAC

values calculated as explained in Ma-

terials and methods. One representative experiment I animal).

I

2

damage induced by DBCP.

i.p. (0. 5 or IO mg/kg), artd killed at 20 min, for DNA

I

0

with 3 animals per exposure (hver* 5 mgikg -

I

h:

Standard errors are shown for the I6 h elution points only.

This indicates that no or very few extra breaks are induced by the preparation procedure, in these slowly replicating cells. The glandular stomach, bone marrow, small and large intestines gave higher values (90-l 70 SSBs per IO”Da). It is possible that these differences are related to the higher replicating activity of the latter tissues. In introductory experiments dose- and time-dependent effects of DBCP in vivo were studied in the liver and kidney; typical elution profiles and their corresponding NAAC values are shown in Fig. I. In both organs SBBs increased with DBCP dose and also with treatment time up to at least 1 h. SSBs were detected in the kidney

G. Br~nborg et al. /Chemico-Biological

2oo

-

200

Interactions IO1 (19%)

39

33-48

LIVER

KIDNEY

LUNG

SPLEEN

BRAIN

BLADDER

TESTIS

BONE

STOMACH

DUODENUM

COLON

MARROW

160

200

2 Q

t

us*

I

1601 m I40

80 l

10 mg/kg,

1H

mglkg,

1H

Sigmficant,

p
J

Fig. 2. Organ-specific DNA damage induced by DBCP. Male Wistar rats were treated with DBCP i.p. (0. IO or 40 mg/kg, i.p.). killed after 1h, and cells/nuclei were prepared from different organs/tissues. Their DNA was assayed for strand breaks with alkaline elution. Mean f S.E.M. are shown. (n) = number of experiments.

at 5 mg/kg as early as 20 min after injection. At the early time points the DNA damage seemed to increase more rapidly in the kidney than in the liver. A dose of IO mg/kg was chosen for most of the subsequent experiments; this dose is below the level which induces renal and testicular necrosis in rats [24,25]. The data in Fig. 2 are pooled from several experiments in which 3-I 1 organs were taken from each rat. Significant DNA SSBs were detected in all the organs tested, but at different frequencies. The bone marrow and large intestine were negative at 10 mgikg DBCP but an increased dose of 40 mg/kg produced SSBs in these tissues also. The glandular (pyloric) stomach showed considerable variations between 5 different samples. Based on the data in Fig. 2 the organs may be ranked according to their net increase in DNA SSBs induced by IO-40 mg/kg DBCP as follows: kidney - duodenum > liver > lung - brain - urinary bladder - glandular stomach > spleen - testis > bone marrow - colon. To study the transplacental genotoxicity of DBCP, female

40

Table

G. Brunborg et al. / Chemico-Biological

Interactions

IO1 (19%)

33-48

I

Transplacenta) Treatment

DNA

damage induced by DBCP DNA

of dam

SSBs (NAAC.

x IO-“h-l) Fetus

Dam DMSO (i)

2.41 f

(ii)

1.49 (I)

0.51 (2)

5.00 f

1.07 (5)

3.89 f

1.61 (3)

2.02 (5)

DBCP (i) 5 mg/kg

13.91 f 0.93 (2)

13.87 f

(i) IO mg/kg

27.58 f

0.59 (2)

30.66 f

7.87 (5)

(ii) 20 mg/kg

107.40 (I)

73.09 f

x.93 (3)

Animals (8 in total) were treated i.p. with 0. 5. IO or 20 mg/kg at day 20 of pregnancy. or 2 dams and from their fetuses (3-5 assayed for DNA

different

SSBs. Values are mean + S.E.M.

thesis: number of independent

Livers from I

livers per dam) were removed 2 h post-exposure

and

(i) and (ii) represent 2 different experiments. In paren-

samples.

rats were dosed at day 20 of gestation with DBCP

at 5, IO or 20 mg/kg, and liver

nuclei were prepared from dams and from several of their fetuses 2 h post-exposure. In the fetal livers dose-dependent DNA damage was detected. and the level of SSBs was the same as in the liver of their mother (Table I). In a separate series of experiments the formation and removal of DNA

damage

during an extended time period was assayed in the liver and the kidney (Fig. 3). Maximum DNA damage induced by IO mg/kg DBCP was recorded at about 4 h; this level

KIDNEY

Time

after

lnpctlon

Fig. 3. Time-dependent

DNA

untreated or pretreated

with PB, were exposed to DBCP

min-3

days), whereafter

(mean f

S.E.M.)

T

(log)

damage in the liver and kidney after DBCP

treatment.

Male rats. either

(IO mg/kg i.p.). killed at different

the liver and kidneys were removed and assayed for DNA

from 3 or more samples. representing at least 2 independent

times (20

damage.

experiments.

Data

G. Brunborg et al. / Chemico-Biological

Interactions

IO1 (19%)

41

33-48

was relatively constant between I and 12 h. By one day the majority of the damage was removed, and DNA SSBs had returned to control levels after 2-3 days. In rats that had been pretreated with PB prior to DBCP, the SSB time kinetics were markedly changed at times later than 20 min postexposure. Particularly in the liver, the maximum level of SSBs was much lower (< IO’%)than in control (not PB-treated) rats, and SSBs returned to control levels at 4 h or earlier. The high persistence of DNA SSBs observed in control rats could reflect the continuous formation of new lesions due to the presence of DBCP; alternatively, DNA repair could be rate limiting. To distinguish between these possibilities, primary hepatocytes were exposed to DBCP in vitro for I h. DBCP was then removed by centrifugation and washing, and cells were further incubated in DBCP-free medium for repair for varying periods of time, after which they were analyzed with alkaline elution. As demonstrated in Fig. 4, DNA strand breaks were apparently removed efficiently during the subsequent incubation period. In addition to frank strand breaks and alkali-labile sites, alkaline elution also detects incisions formed during DNA repair. After DBCP exposure such breaks do not accumulate in substantial amounts in the presence of DNA repair inhibitors (ara Uhydroxyurea) indicating that the major initial lesion(s) are alkali-labile (unpublished observations). The recorded rate of removal of DNA SSBs (Fig. 4) therefore represents net DNA repair. which appears to be 50% complete by - 100 min. This in vitro rate of repair in hepatocytes is fast compared to the removal of SSBs from the liver DNA of control rats (Fig. 3); however, the rate is similar to the comparable time-dependent removal in PB-pretreated animals.

0

45

Fig. 4. Formation

and repair of DNA

with DBCP (O-20 FM) at 37°C for

180

100

Post-exposure

incubation

damage in liver cells in vitro. Primary rat hepatocytes were treated

I

h. DBCP

was then removed by washing. cells were incubated for

O-180 min in medium. and samples were analyzed for DNA representative experiment.

(mini

damage with alkaline elution. Data from

I

42

G. Brunborg et al. / Chemico-3iolagicaI

Inreraciions I01 (19%)

33-48

4. Discussion

The described procedure detects one DNA lesion (strand break) in about 10” nucleotides. This represents a IOOO-fold lower sensitivity compared to the most sensitive alternative assay systems generally suitable for organotropic genotoxicity studies (I adduct per IO’-“’ nucleotide ( j2P postlabelling). or I adduct per IO’-’ (enzyme radioimmunoassay and enzyme immunofluorescence); [26.27]). However. the sensitivity obtained with alkaline elution is still sufficient for genotoxicity screening as well as various mechanistic studies. A main feature of the protocol is a high versatility and capacity. This is achieved by a rapid preparation of nuclei/cells with largely intact DNA from multiple tissues from one or several animals, combined with analysis with a semi-automated elution system. Alternatively, the single-cell gel electrophoresis (comet) assay [28,29] may be used to measure DNA damage in vivo (R. Tice. personal communication); however. few tissues have been studied with the comet method so far [30,31]. Judging from our experience (Fig. 2). most tissues may be analyzed as described. This represents a wider range of tissues compared to earlier use of alkaline elution in vivo [32-341. We have previously demonstrated that the DNA of nuclei taken from the kidney of some species may be degraded during sample preparation; however. this problem seems to have been overcome with the present methodology [ 15,I7,18]. A further advantage is that cells/nuclei are prepared quickly under conditions (low temperature/EDTA) that prevent DNA repair, as opposed to a protocol involving the preparation of single-cell suspensions from in vivo-exposed animals via collagenase/trypsin treatment [35.36]. This is particularly important when studying agents (e.g. MMS. ionizing radiation) inducing DNA lesions that are repaired rapidly. The low level of SSBs induced by DBCP in some tissues (e.g. bone marrow, which is frequently used in in vivo genotoxicity assays) illustrates the usefulness of testing for genotoxicity in more than one or a few cell types. We have recently used the present protocol, combined with i.p. injection of repair inhibitors, to detect DNA damage in epidermal nuclei from mice exposed to IOW doses of ultraviolet radiation [37]. By blocking DNA repair synthesis in vivo a higher sensitivity is achieved and a wider spectrum of DNA modifications may be detected. Considerable efforts have been made in recent years to elucidate the mechanisms of genotoxic and necrogenic effects of DBCP (reviewed in [I I]). The correlation between induced organ toxicity and the DNA damaging potential of a series of deuterated and methylated analogues of DBCP and other evidence led to the hypothesis that DNA damage is an initial event ultimately leading to organ toxicity. P450 conversion of DBCP into 2-bromoacrolein appears to be the predominant activation pathway in bacterial mutagenesis and also plays an important role in DNA damage induction in rat liver cells in vitro [X3]. Experiments with rat testicular cells in vitro and in vivo indicated that DBCP may be activated via GST into DNA-reactive glutathione (GSH) conjugates, probably in the form of episulfonium ions [39-421. This bioactivation pathway appears to be dominating in extrahepatic tissues [I I]. Recently a GSH conjugate of DBCP was reported to alkylate calf thymus DNA with the formation of several N’-guanyl adducts [43]; of the adducts that were detected

G. Brunborg et al. 1 Chemico-Biological

Interactions 101 (19961 33-48

43

in low amounts in DBCP-exposed rat liver, a bis-guanyl adduct may be particularly important in the toxicity of DBCP since it may lead to DNA crosslinks [43]. Recently it was shown that high and multiple oral dosing of mice produces micronuclei in red blood, liver and forestomach cells, but not in cells from the glandular stomach, duodenum, jejunum or caecum [44]. In the present study the I I organs tested were all positive, indicating that DNA damage is a sensitive early marker of DBCP-induced chromosomal mutation. The specific SSB levels recorded (Fig. 2) were not correlated to the P450 content of each organ [45]. The concentration of unmetabolized DBCP in tissues of Wistar rats I h after a single i.p. injection of 20 mg/kg has previously been determined as 135,40 and I I .5 (nmol/g tissue) in the kidney, liver and testis, respectively [25]. Hence there is a direct correlation between the distribution of the parent compound and the level of DNA SSBs in at least some of the organs studied. These levels correlate to some extent with the organ toxicity observed after a single subcutaneous dose of DBCP; all organs were affected with relative sensitivities kidney > testis, lymphohematopoietic tissue. intestine > liver and epididymis [9]. It was concluded that DBCP is a radiomimetic chemical, based on the observation that dividing tissues seemed to have particularly high sensitivities. This may be related to cell-cycle specific DNA repair [46]. Recent flow cytometric studies with thymocytes and HL-60 cells indicate that proliferating cells are particularly susceptible to DBCP which causes selective death of cellsin the S-phase (unpublished data). Since DNA damage appears to be an initiating event in organ toxicity, the degree of atrophy and cell death in the various organs would be expected to be influenced also by their rates of cell proliferation. An intermediate level of DBCP-induced SSBs in the brain coincides with a distribution of the non-polar compound into this organ. In the fetal liver the number of SSBs was not significantly different from that in the liver of the exposed dam (Table 1). DBCP is apparently transferred across the placenta and activated in the fetal liver. Conjugation via GSTs is likely to contribute to this activation, since the fetal liver contains substantial amounts of GSTs towards the end of pregnancy [47], whereas the P450 levels are very low [48]. CYP2EI, which is the most likely candidate for P450-bioactivation of DBCP [49], is not detected in the fetal rat liver [50]. Environmental exposure to a number of chemicals may lead to exposure of the fetus [51] but most often the fetal tissue is less affected than that of the mother [52]. In contrast, the present data with DBCP indicates that this compound is potentially as genotoxic to the fetus as to the dam. There are no published data on the transplacental carcinogenicity of DBCP. In a teratogenicity study with mice [53] and rats [54], no increase in the skeletal or visceral abnormalities were observed in the fetuses of DBCP-treated dams. However, in adult offspring intratesticular testosterone and luteinizing hormone receptor concentrations, as well as absolute and relative testis weights, were severely reduced in all groups of rats exposed to DBCP during fetal life [IO], and histological examination of their testes revealed damage in the seminiferous tubules and in germinal cells. The terminal half-life of DBCP in rats is 2.3-3.6 h (referenced in [I I]). DBCP is eliminated from organs such as the liver and kidney of treated rats [55], corresponding to the persistent DNA damage detected in these organs (Fig. 3). In rats

44

G. Brunborg et al. / Chemico-BiatogiealInteractions IO1 (19%) 33-48

pretreated with PB, which is known to reduce the toxicity of DBCP [56], DBCP is removed more rapidly from tissues probably due to an increased hepatic detoxification (551. This is paralleled with a more efficient removal of DBCP-induced DNA SSBs from the liver and kidney of PB-pretreated rats (Fig. 3). It appears that this removal of SSBs is comparable to the rate of DNA repair observed in cultured hepatocytes upon incubation in DBCP-free medium (Fig. 4). From Fig. 3 it was calculated that the time-integrated DNA damage (0- 12 h) is reduced by PB pretreatment, relative to control, to 3% in the liver, and 28% in the kidney. Previously we have shown that PB pretreatment totally abolishes the kidney necrosis induced by 40 mg/kg DBCP i.p. [55]. Taken together, the data indicate that the distribution characteristics of DBCP are of major importance for the extent of DBCP-induced DNA damage and its persistence in various organs of rats. A further key factor affecting DBCP-induced DNA damage is the cell type specific metabolic activation, as demonstrated by in vitro studies. P450-dependent activation product(s) of DBCP are partly diffusible, whereas the reactive GSH-dependent metabolite(s) are short-lived and apparently do not penetrate the cell membrane [38,57]. In addition to the tissue distribution of DBCP, the degree of initial DNA damage would be expected to depend primarily on the local metabolizing capacity of the tissue if GSH/GST activation is essential. In the rat, the GST activity (with I-chloro-2,4-dinitrobenzene as substrate) is highest in the liver, followed by the testis (67%) and the kidney (19%) [58]. The metabolic conversion of DBCP has recently been studied in isolated liver, kidney and testicular cells, and with various purified human and rat GST isozymes; the data indicate that the rate of formation of DBCP water soluble metabolites depends on the specific isozyme composition of the tissue [57]. It has recently been reported that DBCP produces tumors (mostly hepatocellular carcinomas) in the fish Danio rerio [44]. Carcinogenicity studies with mice and rats gave a high incidence of squamous cell carcinomas in the forestomach and mammary gland adenocarcinomas in female rats, at the lowest oral dose of 15 mg/kg, 5 days per week [59]. Most (about 90%) of the animals died from their tumors at 70-80 weeks. An inhalation study [60] demonstrated nasal and lung tumor induction. A high DBCP-dependent mortality in these studies may have prohibited the development of tumors in other target tissues at lower doses of DBCP. Most probably, however, the development of DBCP-induced tumors depends on other factors in addition to the initial level of SSBs. One contributing factor may be DNA repair, and the present methodology appears to be well-suited to study rates of DNA repair in various target tissues. Acknowledgements

We thank Bente Trygg, Kirsti Haug and Solfrid Hegstad Nyholm for excellent technical assistance, and Dr. Arne Mikalsen for helpful suggestions. This project was supported by NIH Grant ES 02728 and by the Norwegian Research Council.

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