Critical role of oxidative stress in estrogen-induced carcinogenesis

Abstract

Mechanisms of estrogen-induced tumorigenesis in the target organ are not well understood. It has been suggested that oxidative stress resulting from metabolic activation of carcinogenic estrogens plays a critical role in estrogen-induced carcinogenesis. We tested this hypothesis by using an estrogen-induced hamster renal tumor model, a well established animal model of hormonal carcinogenesis. Hamsters were implanted with 17beta-estradiol (betaE2), 17alpha-estradiol (alphaE2), 17alpha-ethinylestradiol (alphaEE), menadione, a combination of alphaE2 and alphaEE, or a combination of alphaEE and menadione for 7 months. The group treated with betaE2 developed target organ specific kidney tumors. The kidneys of hamsters treated with alphaE2, alphaEE, or menadione alone did not show any gross evidence of tumor. Kidneys of hamsters treated with a combination of alphaE2 and alphaEE showed early signs of proliferation in the interstitial cells. Kidneys of hamsters treated with a combination of menadione and alphaEE showed foci of tumor with congested tubules and atrophic glomeruli. betaE2-treated tumor-bearing kidneys showed >2-fold increase in 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) levels compared with untreated controls. Kidneys of hamsters treated with a combination of menadione and alphaEE showed increased 8-iso-PGF(2alpha) levels compared with untreated controls, whereas no increase in 8-iso-PGF(2alpha) was detected in kidneys of alphaEE-treated group. A chemical known to produce oxidative stress or a potent estrogen with poor ability to produce oxidative stress, were nontumorigenic in hamsters, when given as single agents, but induced renal tumors, when given together. Thus, these data provide evidence that oxidant stress plays a crucial role in estrogen-induced carcinogenesis.

Figure 1

Paraffin section of an untreated male Syrian hamster kidney stained with hematoxylin and eosin. Normal kidney architecture with normal convoluted tubules (CT) and glomerulus (G) is observed. Magnification = ×40.

Figure 2

Paraffin section of a tumor-bearing kidney stained with hematoxylin and eosin. The tumors were induced by treatment of male Syrian hamsters with βE2 for 7 months. (a) An abnormal kidney architecture with tumor nodules (T) and congestion of scattered convoluted tubules (arrow) within the tumor nodules can be observed. (b and c) The tumor nodules are composed of a combination of round to spindled hyperchromatic cells (b, arrows), and in some of the tumor nodules (T), entrapped and atrophic glomeruli (G) are present (c). Many of the congested tubules (Tb) are filled with pink eosinophilic deposits (arrow in c, arrowhead in d) and are lined by somewhat flattened epithelial cells (d, arrow). Magnification: a = ×4; b = ×40; c = ×10; d = ×40.

Figure 3

Semiquantitative estimate of the total kidney damage in hamsters treated with 25-mg pellets of βE2, αE2, αEE, menadione, a combination of αE2 + αEE, or a combination of αEE + menadione for 7 months as s.c. implants. The control group was sham operated and received no estrogen implants. Hematoxylin and eosin-stained sections of paraffin-embedded tissues were analyzed microscopically for the extent of renal damage as described in Materials and Methods. Increased congestion of the convoluted tubules (c-ct) and glomeruli (c-g) was observed in kidneys of hamsters treated with βE2, menadione, a combination of αE2 + αEE, or a combination of αEE + menadione. Glomerular atrophy (a-g) and proliferation (p) is observed in kidney tissue of the groups treated with βE2 or a combination of αEE + menadione for 7 months. 1, untreated control; 2, αE2; 3, αEE; 4, αE2 + αEE; 5, menadione; 6, menadione + αEE; 7, βE2. a, P < 0.05 for both c-g and c-ct when compared with untreated, αE2-treated, or αEE-treated hamster kidney by an unpaired t test. b and c, P < 0.05 for a-g and p, respectively, when compared with any other group by an unpaired t test. For each type of damage, six to eight hematoxylin and eosin-stained kidney sections were examined from each treatment group and scored, and data are expressed as a mean of the individual scores. The SEM for all of the four types of kidney damage (c-ct, c-g, a-g, and p) in all of the seven different treatment groups varied from 0% to 22% except for c-ct for the group that was treated with αE2, where the % error was 64. This group showed very little kidney damage.

Figure 4

Paraffin section of a male hamster kidney stained with hematoxylin and eosin. Male Syrian hamsters were treated with a combination of αEE and αE2 for 7 months. (a) Vascular congestion of glomeruli (G) and convoluted tubules (CT) is observed. (b) Kidneys contain foci where there are atypical collections of both interstitial cells (arrow) and CT. (c) Some of the tubules contain cuboidal cells that have scant cytoplasm and irregular, slightly pleomorphic hyperchromatic nuclei (arrowheads). (d) Some slightly crowded renal collecting tubules (arrows) are also observed in kidneys of hamsters treated with a combination of αEE and αE2 for 7 months. Magnification: a–c = ×40; d = ×10.

Figure 5

Paraffin section of a tumor-bearing kidney stained with hematoxylin and eosin. The tumors were induced by treatment of male Syrian hamsters with a combination of αEE and menadione for 7 months. (a) Sections of the kidney demonstrate foci of tumor (T) with congested renal tubules (Tb and arrowheads). (b) The tumor is comprised of hyperchromatic cells with nuclear crowding (arrows). Congested renal tubules (arrowheads) and atrophied glomeruli (G) are also seen entrapped within the tumor nodule. Magnification: a = ×10; b = ×40.

Figure 6

8-iso-PGF_2α levels in kidney homogenates of hamsters treated with αE2, βE2, αEE, menadione, αE2 + αEE, or menadione + αEE for 7 months. 8-iso-PGF_2α was quantified by using a commercially available kit from Assay Designs as described in Materials and Methods. A >2-fold increase in 8-iso-PGF_2α is detected in βE2-treated tumor-bearing kidneys of hamsters. The fold increases in 8-iso-PGF_2α are 1.38, 1.50, 1.52, and 1.55, respectively, for kidneys of hamsters treated with αE2, menadione, αE2 + αEE, and menadione + αEE compared with untreated controls. 8-iso-PGF_2α and protein were analyzed from 10 kidney homogenates from each group, and data are expressed as mean 8-iso-PGF_2α pg/mg protein ± SEM. *, P < 0.05 compared with untreated controls by an unpaired t test; **, P < 0.05 compared with αEE treated group by an unpaired t test.