Diethylstilbestrol liver carcinogenicity and modification of DNA in rats

Cancer Letters, 68 (1993) 193- 198 Elsevier Scientific Publishers Ireland Ltd.

Diethylstilbestrol rats

193

liver carcinogenicity

and modification

of DNA in

Gary M. Williams, Michael Iatropoulos, Raymond Cheung, Leila Radi and C.X. Wang American Health Foundation,

One Dana Road, Valhalla, NY 10595 (USA)

(Received 6 August 1992) (Revision received 3 December 1992) (Accepted 4 December 1992)

Administration of diethylstilbestrol to female Sprague- Dawley rats at IO mg/kg body weight daily by gavage for 1 year induced liver adenomas adenomas.

and

carcinomas

and

pituitary

Using the 32P-postlabeling assay for DNA alterations, at 24 h after administration of a single dose of 100 mg/kg, modified bases were found. Keywords:

diethylstilbestrol; liver carcinogenicity; liver DNA adducts; rats Introduction

The diphenylethylene compound diethylstilbestrol (DES) is a human and a rodent carIn utero exposure cinogen [7,8,10,15]. resulted in increases in cervical, vaginal, mammary, ovarian and lymphoid tumors in mice [lo, 151 and vaginal neoplasms in rats [l]. Chronic administration to adult mice resulted in cervical and endometrial adenocarcinomas, mammary adenocarcinomas, osteosarcomas, and mesotheliomas [8]. Exposed rats displayed pituitary and mammary tumors [Zl] Correspondence

Foundation,

to:

Valhalla,

0304-3835/93/$06.00 Printed and Published

Gary M. Williams, M.D., American NY 10595,

U.S.A.

Health

(914)789-7138

0 1993 Elsevier Scientific Publishers in Ireland

and in one study with castrated male WF rats, liver tumors were produced [24]. Syrian hamsters developed tumors of the uterus, cervix, vagina, pituitary, testes and kidney [12,23] and hepatocellular carcinomas were produced in European hamsters [23] and in Armenian hamsters [2]. In spite of the plethora of carcinogenic effects of DES, it is often referred to as only a ‘promoter’ of neoplasia [27], as is the structurally related triphenylethylene drug tamoxifen [3], although it is also carcinogenic [ 171. This laboratory recently reported the marked hepatocarcinogenicity in female rats of tamoxifen [17]. Because of the structural similarity of DES to tamoxifen and in order to extend the data base on DES, we studied DES for hepatocarcinogenicity in female rats using the same protocol as for tamoxifen. Also, in light of reports that DES induced DNA modifications in hamster tissues [6] we assayed for DNA alterations in rat liver. We now report that in female rats, DES is hepatocarcinogenic within 1 year and causes liver DNA alterations. Materials

and Methods

One hundred and ten female SpragueDawley rats (Crl:CD(BR): Charles River, Kingston, NY), divided into two groups, were employed in the chronic (l-year) study. Half of them were kept as controls and half received Ireland Ltd.

194

diethylstilbestrol (DES), obtained from Sigma (Sigma No. D-4628) Chemical Co. St. Louis, MO, USA. The Lot No. was 47F-0103. The carrier substance was carboxymethylcellulose (CMC), obtained from Sigma (Sigma No. C8758). The Lot No. was 19F-0022. A daily dose of 10 mg/kg body weight was administered by gavage, with the volume remaining constant at 1 ml/rat. The protocol for this study was approved by the Institutional Animal Care and Utilization Committee. Standard Operating Procedures for Good Laboratory Practice Regulations [26] were used for all procedures. Each rat was assigned a study animal number, on selection of animals by body weight for test groups. The animals were identified by an ear notch and toe clipping and were housed 3 in a cage with sawdust bedding in solid floor cages in temperature and humidity controlled rooms. The bedding was the standard ‘Beta Chip’ hardwood rodent variety (no cedar was used). Twelve hours of continuous low level fluorescent lighting (5 ft candles/ft2) was provided daily. The NIH-07 diet and tap water were provided ad libitum. The certified diet and bedding had a certificate of analysis for standard contaminants. Diet, tap water and bedding was analyzed. The facility is accredited by the American Association for Accreditation of Laboratory Animal Care, and is an institutional member of the American Association for Laboratory Animals Science. The care of animals conformed to the NIH Guide for the Care and Use of Laboratory Animals [25]. A physical examination was conducted 4 days before the study started and every 2 months to the end of the study. The animals were observed daily for their general condition and behavior. The body weights were measured weekly for the first 8 weeks, every 2 weeks for weeks 9 through 16 and monthly thereafter until the end of the study. Animals were killed at 1, 3, 6 and 12 months. The 35 rats killed at 1 and 3 months were used for biochemical studies [ll]. Ten rats/group were available at 6 and 12 months

for pathology. At these times, the rats were weighed and subsequently euthanized with CO2 gas. All animals received a complete macroscopic examination. At necropsy all external surfaces, all orifices, carcass, cranial cavity, external surfaces of the brain and spinal cord and the cut surface of the spinal cord, the nasal cavity and paranasal sinuses, the thoracic, abdominal and pelvic cavities and their viscera as well as the cervical tissues and organs were examined and findings recorded. The liver and the following tissues were sampled and preserved in neutral buffered 10% formalin (NBF): femoral muscle, muscle from the abdominal wall, diaphragm, skin, mammary gland, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, liver, pancreas, kidneys, ureters, urinary bladder, urethra, ovaries, uterus, vagina, thymus, spleen, mesenteric lymphnodes, adrenal glands, pituitaries, thyroids/parathyroids, brain, spinal cord, sciatic nerve, eyes and grossly observed lesions. Liver, adrenals, ovaries, uterus, mammary glands, eyes, bone and all grossly observed lesions were embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E) and examined microscopically. Statistical evaluation was performed utilizing the Student t-test analysis. It was applied to body weights, and incidence of pertinent microscopic findings. For the study of effects of DES on liver DNA, 14-month-old female rats of the same strain and source were used. Three rats were given 100 mg/kg DES by intraperitoneal (i.p.) injection and one served as the vehicle control. DES, Lot No. 02523HT was obtained from Aldrich Chemical Co., Milwaukee, WI, USA. It was dissolved in olive oil (Bert011Inc., Lot No: ITA/853NA/35, Secaucus, NJ, USA) at a concentration of 25 mg/ml. The animals were killed by decapitation 24 h after dosing. DNA was extracted from the livers using a modified Marmur method [5] and modified bases were analyzed by the 32P-postlabeling method of Randerath [22]. The extracted DNA samples were enzymatically digested, labeled with 32P and treated with nuclease Pi

195

Table 1.

Mean body weights in female rats exposed

to diethylstilbestrol

(DES)for up

to

12 months.

Group

Initial

l-Month

S-Month sacrifice

6-Month sacrifice

12-Month sacrifice

Control mean * S.D. DES mean zt S.D.

263.0 5.2 262.0 4.8

288.0 10.4 206.0’ 20.3

342.5 55.4 217.9’ 30.4

402.3 52.7 218.4’ 13.7

510.0 108.6 235.4’ 31.4

‘Statistically

significant from controls

at P < 0.01.

the following modifications: carrier-free 10 mCi/ml [32P]phosphate with an activity __ of . (Amersham Co., Arlington Heights, IL, USA) was used and the labeled adducts were separated on polyetheneimine (PEI) cellulose plates using the following solvents, Dl: 1 M sodium phosphate (pH 6.8), D3: 3.8 M lithium formate/ M urea (pH 3.4) and D4: 0.7 M sodium phosphate/7 M urea (pH 6.8). The labeled adducts were located by autoradiography for 16 h at - 80°C using DuPont Cronex-4 X-ray film and DuPont Lightning Plus intensifier screens. Adducts were evaluated qualitatively from up to 10 pg DNA as autoradiographic spots. The average concentration was 2.2 jkg/ml.

with

Results

The compound-related mortality was 20%. Most of the early deaths were due to gavage accidents, while later some were due to liver tumors. There was a dramatic decrease in Table II. Exposure time

Liver neoplastic Group

effects of diethylstilbestrol No. of rats

DES” DES

10 10

“Dose of 10 mg/kg/day. bNumber of change over number

(DES) in female rats exposed

Adenomas Incidence

6 months 12 months

mean body weights of the DES group (Table I). This decrease was evident at 1 month and remained statistically significant for the duration of the study. Administration of DES for 6 months caused hepatocellular adenomas in a 10% incidence (Table II). By 12 months there was a 30% incidence of hepatocellular carcinomas. The carcinomas were massive and multifocal replacing most of the parenchyma in most liver lobes. Estrogen target sites (organ/tissues) displayed time-related changes, with increases in proliferative and atrophic changes (Table III). Specifically, there was reduced number of corpora lutea, increased number of atretic follicles and increased number and dilation of secondary follicles with atrophy in the ovaries of DESexposed animals. Furthermore, there was glandular atrophy and endometrial hyperplasia in the uteri of the DES group. In the mammary glands of the DES group there was acinar atrophy, with multifocal areas of hyperplasia. Finally, in 20% of the DES animals,

for up to 12 months.

Carcinomas

(46)

10 10

of animals with the change.

Multiplicityb

Incidence

l/l

0 30

2/l

(%,)

Multiplicity 0 5/3

196

Table111.Summary of pertinent microscopic findings in organs other than liver in female rats exposed diethylstilbestrol (DES) for up to 12 months. Microscopic

findings

Ovary

Reduced number of corpora lutea Increased number of atretic follicles Cystic dilation of follicles Uterus Glandular atrophy Immature endometrial surface epithelium Endometrial hyperplasia Mammary gland Acinar atrophy and focal hyperplasia (activation) Pituitary

Adenoma

Control

to

DES

WNL WNL 2 (20%)” 4(40%) WNL 2(20%) WNL 2(20%) WNL

Fig.1. 32P-Past-labeling

3(30%) WNL 2(20%)

DES = Dose of 10 mg/kg/day. WNL = Within normal limits. 1 = Minimal; 2 = slight; 3 = moderate; 4 = severe change. aPercent incidence in parenthesis, the number of rats per group was 10.

chromophobe adenomas were present in the pituitaries. In the 32P-postlabeling analysis, no spots were detected in autoradiograms of control liver DNA samples. In the livers of the DES group (100 mg/kg), 6 - 7 spots were evident (Fig. 1). Discussion This study has demonstrated for the first time that DES can induce liver tumors in female rats and give rise to modifications in liver DNA. DES binds to the estrogen receptor [ll], which is present in rat liver [4,11], and exerts an estrogenic effect in the liver [ 111. DES has

analysis of liver DNA from 14-month-female Sprague Dawley rats given a single intraperitoneal dose of 100 mg/kg of DES and killed 24 h after dosing. Adducts are lettered to the right of each spot. Seven spots (a -3) representing DES adducts are evident.

been presumed to be a liver neoplasm promoter acting through hormonal effects [27]. However, the current finding of liver carcinogenicity beginning at 6 months and evident by 12 months of exposure in the incidence of 30%) strongly suggests that DES is capable of initiating the neoplastic process in the liver, in agreement with the conclusion of Sumi et al. [24]. In a study with tamoxifen, which is structurally related, we found that it was also hepatocarcinogenic under similar conditions [17] whereas another structurally related compound toremifene was not, although it displays comparable antiestrogenic/estrogenic effects in the liver [ll]. Moreover, Han and Liehr [9] have reported the formation of DNA adducts by tamoxifen. Thus, estrogenic action does not appear to be the basis for the liver carcinogenicity of these polyphenylethylene compounds. Metzler et al. [20] have provided evidence that DES can be activated at the ethylene double bound to reactive species. Positive findings have been made with in vitro systems [l&19],

197

although it was negative in the rat hepatocyte DNA repair test [ZS]. The production of DNA alteration by DES reported by Gladek and Liehr [6] and observed in this study support the possibility of DNA-reactivity of DES. The adducts, however, could also arise from an endogenous DNA-binding by-product, as has been suggested in the case of diverse estrogens which give rise to similar DNA modifications [13]. Regardless, the present findings extend those of Liehr who reported induction of DNA modifications in hamster kidney, which is highly susceptible to the carcinogenic effects of DES [E]. It remains to be established whether DES or tamoxifen can alter genes critical to neoplastic conversion. Nevertheless, the carcinogenicity of these agents may well depend upon DNA alteration and their potency may reside in a combination of genotoxic and nongenotoxic effects, as for many other carcinogens [29].

9

10

11

12

13 14

15

16

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