Critical role of oxidative stress in estrogen-induced carcinogenesis (VSports最新版本)

VSports - Treatment of Animals.

Male Syrian hamsters (4–6 weeks old; Harlan Sprague–Dawley, Madison, WI) were housed in our animal facility with Purina rodent chow and water available ad libitum throughout the experiment. Hamsters were implanted s. c. with 25-mg pellets of βE2, αE2, αEE, menadione, a combination of αE2 + αEE, or a combination of αEE + menadione. These hamsters received a second estrogen or menadione pellet 3 months after initial treatment. Before implantation of the drug pellets, hamsters were anesthetized with a combination of ketamine and xylazine (ketamine, 100 mg/kg body weight, i. p. , and xylazine, 10 mg/kg body weight, i. p. ). Estrogen and menadione pellets were prepared by using a hand press and implanted into the hamsters s. c. as described (10, 12). There were 10 hamsters in each group. A control group of 10 animals was sham operated and left untreated. Hamsters were killed after 7 months and inspected macroscopically for tumor nodules on the surface of each kidney as reported (31). Portions of liver and kidney tissue were placed on dry ice and stored at −80°C for further studies. Other portions of liver and kidney were placed in 10% buffered formalin for histopathological evaluation and immunocytochemical studies. Menadione and all estrogens used in the present study were purchased from Sigma VSports.

"VSports最新版本" Tissue Preparation for Histopathology and Immunocytochemistry.

The formalin-fixed tissue was embedded in paraffin, and sections of 4- to 5-μm thickness were cut VSports app下载. Paraffin-embedded sections of the kidneys and livers were stained with hematoxylin and eosin for histopathological evaluation. Gross examination and histological sections were interpreted by two independent pathologists in a blinded fashion, without knowledge as to how the animals were stratified. Paraffin-embedded sections were also used for cell-specific expression of estrogen receptor (ER)-α and -β proteins. Deparaffinized sections were incubated with the corresponding primary antibodies: ER-α (Santa Cruz Biotechnology, SC-542) and ER-β (Santa Cruz Biotechnology, SC-6821) at dilutions suggested by the suppliers. Incubation with the primary antibodies was performed overnight at 4°C. Nonspecific sites were blocked by covering sections with solutions of 1% BSA (Sigma). After washing in PBS, the sections were incubated with peroxidase-conjugated, affinity-purified F(ab′)2 fragment of donkey anti-rabbit IgG (Jackson ImmunoResearch) for 60 min at room temperature as described (12). Slides were rinsed three times in PBS, and staining was developed by incubating with 3,3′-diaminobenzidine tetrachloride/H2O2 (Sigma) for 2–5 min. 3,3′-Diaminobenzidine solution was prepared according to the manufacturer's recommendations. After staining, the sections were rinsed in distilled water, dehydrated in ethanol/water baths with decreasing water content, and finally rinsed in xylene before being mounted with a permanent mounting medium.

Semiquantitative Analysis of Critical Histopathological Differences.

The severity of the total kidney damage was evaluated by a scoring system that gave a semiquantitative measurement of the damage as described (32). The four types of damage for which semiquantitative analysis was performed were glomerular atrophy, glomerular congestion, tubular congestion, and nodular proliferation. One hundred nephrons (glomeruli and adjacent tubules) were examined histologically, and each nephron was scored from 0 to 4. For glomerular atrophy, glomeruli that contained >20 nuclei were graded as “0,” and those glomeruli which contained <10 nuclei were scored as “4. ” For glomerular congestion, glomeruli that did not contain eosionophilic and pink deposits were graded as “0,” and those that had ≈80% of glomeruli with such deposits were graded as “4 V体育官网. ” For tubular congestion, convoluted tubules that did not contain eosinophilic and pink deposits were graded as “0,” and those convoluted tubules that contained ≈ 80% deposits were graded as “4. ” Intermediate stages were graded as 1, 2, and 3. For proliferation, sections of kidneys were graded as “4” if they contained tumor nodules, and as “0” if they did not have any tumor nodules. The sum of individual scores of 100 counts on each kidney section was used as an estimate of the severity of the kidney damage. A total score of 1,600 was given by the sum of the four descriptive damages described above (each kind of damage getting a maximum score of 400). For each type of damage, six to eight hematoxylin and eosin-stained kidney sections were examined from each treatment group and scored, and data were expressed as a mean ± SEM of the individual scores.

Analysis of 8-iso-PGF_2α Levels.

Total 8-iso-PGF_2α levels in kidney tissue of hamsters treated with different chemicals were quantified by using a direct 8-iso-PGF_2α enzyme immunoassay kit from Assay Designs (Ann Arbor, MI, catalog no. 900-091) according to the supplier's recommendations. Kidney tissue (50–100 mg) was homogenized in cold PBS (pH 7.4) containing 0.005% butylated hydroxytoluene. Tissue homogenates (10% wt/vol) were prepared by using a PRO 200 homogenizer with a 5 mm × 75 mm generator (PRO Scientific, Oxford, CT) in 2-ml microfuge tubes. Homogenization was carried out by moving the motor speed dial of the homogenizer from 0 to 5 (0–30,000 rpm) back and forth five times with a total homogenization time of ≈5 s. 8-iso-PGF_2α esters in 100 μl of the total kidney homogenate were hydrolyzed by incubation with 25 μl of 10 n NaOH at 45°C for 2 h. The reaction mixture was cooled on ice for 5 min and neutralized with 25 μl of 12 N HCl, and centrifuged in a microcentrifuge for 5 min. The clear neutralized supernatant was transferred into a new microfuge tube, and 50 μl of the neutralized sample was used for 8-iso-PGF_2α assay. The samples were incubated with the 8-iso-PGF_2α antibody for 18 h at 4°C in a 96-well format. After incubation, the contents of the wells were emptied and washed with wash buffer; wash buffer was removed from the wells, and the color was developed by incubation with 200 μl of p-nitrophenyl phosphate for 45 min at room temperature. The reaction was stopped by the addition of 50 μl of stop solution, and the plate was read at 405 nm. A standard curve was generated by measuring the optical density of 160–100,000 pg/ml of 8-iso-PGF_2α standards that were processed simultaneously with unknown samples on the same plate. Protein concentrations from the neutralized homogenates were determined by using a Pierce protein assay kit. 8-iso-PGF_2α and protein were analyzed from 10 kidney homogenates from each group, and data were expressed as mean 8-iso-PGF_2α pg/mg protein ± SEM.

Statistical Analysis.

Statistical analysis was performed by using spss statistical software package (SPSS, Chicago). Unpaired t test was used to assess significance between the two different treatment groups. Tumor incidence was analyzed by Fisher's exact test.

Tumor Incidence.

On macroscopic examination, no tumor nodules were detected in untreated hamsters and in groups of hamsters that were treated with αE2, αEE, menadione, or a combination of αE2 and αEE for 7 months (Table 1). In contrast, hamsters treated with either βE2 or a combination of αEE and menadione for 7 months developed renal tumors. The tumor incidence was 90% for the group treated with βE2 and 30% for the group treated with a combination of αEE and menadione (Table 1). The mean number of tumor nodules was higher in the group treated with βE2 compared with the group treated with a combination of αEE and menadione (Table 1).

Histopathological Analysis.

Sections from untreated hamster kidneys demonstrated normal convoluted tubules and glomeruli within the cortex (Fig. 1). The corticomedullary junction appeared normal, as did the renal hilum, which was lined by normal transitional urothelium.

Kidneys of hamsters treated with βE2 for 7 months were abnormal and contained numerous tumor nodules (Fig. 2a). The nodules were composed of a combination of round hyperchromatic cells and round to spindled, irregular, hyperchromatic cells (Fig. 2b). Scattered tubules within the tumor nodules were congested (Fig. 2a). Most of the tumor nodules were in the cortical area of the kidney and, in some of the nodules, entrapped and atrophic glomeruli were seen (Fig. 2c). Sections from the kidney away from the tumor were also markedly abnormal and demonstrated large dilated congested convoluted tubules, which were lined by somewhat flattened, cuboidal epithelial cells (Fig. 2d). Many of the congested tubules were filled with pink eosinophilic deposits (Fig. 2d). A significant increase in the number of congested convoluted tubules, congested glomeruli, atrophic glomeruli, and nodular proliferation was detected in the βE2-treated tumor-bearing kidneys compared with untreated controls by using the semiquantitative estimates of the total kidney damage (Fig. 3).

Microscopic evidence of tumor nodules was not seen in any of the kidneys of hamsters treated with αE2 for 7 months. Kidney sections showed normal glomeruli and convoluted tubules within the renal cortex. No abnormal proliferations were seen. The medulla and hilum were microscopically normal (data not shown).

Atypical congested convoluted tubules were seen in kidneys of hamsters treated with αEE for 7 months (Fig. 3). In some areas, the convoluted tubules were lined by crowded hyperchromatic cuboidal cells, which had decreased cytoplasm, and the kidney sections also showed some congested glomeruli (see Fig. 7, which is published as supporting information on the PNAS web site, www.pnas.org). No nodular proliferation was detected in the kidney sections of hamsters treated with αEE for 7 months (Fig. 3).

Kidney sections of hamsters treated with menadione for 7 months demonstrated renal convoluted tubules with evidence of vascular as well as glomerular congestion (Fig. 3) and the presence of an altered epithelial layer of convoluted tubules (see Fig. 8, which is published as supporting information on the PNAS web site). No tumor nodules were identified, and there was no evidence of atrophic glomeruli (Fig. 3).

On microscopic examination, vascular congestion of glomeruli and of convoluted tubules was observed in the kidneys of hamsters treated with a combination of αE2 plus αEE for 7 months (Figs. 3 and 4a). Kidneys contained foci where there were atypical collections of both interstitial cells and convoluted tubules (Fig. 4b). In these areas, some of the tubules contained cuboidal cells, which had scant cytoplasm and irregular, slightly pleomorphic, hyperchromatic nuclei (Fig. 4c). Some slightly crowded hyperchromatic renal collecting tubules were observed (Fig. 4d).

Kidney sections of hamsters treated with a combination of αEE plus menadione demonstrated foci of tumor that contained congested tubules (Fig. 5a). The tumor nodules were characterized by hyperchromatic, spindled to rounded cells with marked nuclear crowding and overlapping (Fig. 5b). This group showed significant congestion of both renal convoluted tubules and glomeruli when compared with αE2 and αEE treated groups (Fig. 3). Glomerular atrophy and nodular proliferation were detected in the kidneys of hamsters treated either with a combination of αEE plus menadione, or with βE2 for 7 months (Figs. 3 and 5b).

Microscopic examination of tissue sections from livers of hamsters treated with βE2, αE2, αEE, menadione, αE2 + αEE, or menadione + αEE did not show any evidence of tumor or dysplasia (data not shown).

Immunocytochemical Analysis.

Renal tumors induced by a carcinogenic estrogen or by a combination of a noncarcinogenic estrogen and a chemical known to produce oxidative stress were characterized immunocytochemically. Cell-specific expression of ER-α and -β was examined in the kidneys of hamsters treated with different estrogens, either alone or in combination with menadione. ER-β protein expression was not detected either in control or in tumor-bearing kidneys (data not shown). A very poor immunoreactivity for ER-α protein was observed in the tubules of untreated control kidneys (see Fig. 9, which is published as supporting information on the PNAS web site). Intense nuclear staining for ER-α protein was found in βE2-induced renal tumors (see Fig. 9). ER-α protein expression was not detected in the glomeruli or kidney tubules of menadione-treated animals (data not shown). Moderate to strong immunoreactivity for ER-α protein was observed in the renal tumor area of αEE plus menadione-treated hamsters and some of the tubules within the tumor mass also expressed high levels of ER-α protein (see Fig. 9).

8-iso-PGF_2α Analysis.

Total 8-iso-PGF_2α levels in kidney tissue of hamsters treated with different chemicals were quantified. A >2-fold increase in 8-iso-PGF_2α was detected in tumor-bearing kidney homogenates of hamsters treated with βE2 for 7 months compared with untreated controls (Fig. 6). Kidney homogenates of hamsters treated with αEE for 7 months did not show any increase in 8-iso-PGF_2α levels compared with untreated controls (Fig. 6). The fold increases in 8-iso-PGF_2α were 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 (Fig. 6). There were no significant differences in 8-iso-PGF_2α levels between αE2-, menadione-, αE2 + αEE-, and menadione + αEE-treated groups.