Results

Enzyme Assays. Effects of MG and liver cytosol on antioxidant enzyme activities are depicted in Figure 1. In general, specific activities were notably higher in liver compared to MG ranging from- 6.0-fold higher for NQOl to 100.0-fold higher for CAT. E2-treatment for 6 and 12 w increased hepatic NQOl activity by 4.1- and 3.7-fold, respectively (Figure 1B); while GST increased by 1.5- and 2.0-fold, respectively (Figure 1D). Both activities returned to control levels by week 28 (Figure 1B, D). Hepatic GPx activity did not change significantly over the course of the treatment (Figure 1, F); however, its activity increased significantly in MG tissue at 28 w (Figure 1E). CAT activity decreased in liver and MG tissue at 28 w (Figure 1G, H).

NQOl GST CP* CAT

NQOl GST CP* CAT

Figure 1. Antioxidant enzyme activities in MG and liver of E2-treated ACI rats Data represent the mean ± SE of 3 rats/group. Data from control animals (C) at different periods were similar and pooled together (n = 9). Statistically different from C.

SULT1A1 activity was reduced markedly (95%) after E2 treatment (Figure 2), and preceded the appearance of MG tumors.

Figure 2. MG and liver SULT1A1 activity of E2-tteated ACI rats. Data represent the mean ± S.E. of 3rats/group. Data for control animals (C) at different periods were similar and pooled together (n = 9). *Statistically different from C.

SULT activity was measured using PNP, E2, and DHEA as substrates targeting activities of SULT1A1, -E1, and -2A1, respectively. Kinetic parameters for liver and MG SULTs activities are described in Table 1.

Table 1. Kinetic Parameters of E2-metabolizing Enzymes in MG and Liver of ACI Rat.

Kinetic Parameters

Table 1. Kinetic Parameters of E2-metabolizing Enzymes in MG and Liver of ACI Rat.

Kinetic Parameters

Enzyme System

Substrate

Tissue

Km <liM)

Vmax (pM/mg/min)

PNI1

Liver

2

6700

SULT t Al

MG

2.1

1.975

Liver

10

5040

Sulfotransferase

MG

48

6.97

DHEA

Liver

10.8

1860

SULT2A1

MG

Linear to 100 ^iM

nd

Liver microsomal NADPH-dependent oxidation of E2 formedmajoritary 2-OH-E2 and estrone (Ei) and small amounts of 4-OH-E2 and 2-OH-E] as reported previously (23, 26) (Table 2). E2 exposure caused a modest increased in E! formation (~ 1.5-fold). In MG microsomes, several oxidized derivatives such as 6a-, 16a-, 16(J- and 2-OH-E2, and 6-keto- and 2-OH-Ei, and E| were detected in small amounts (data not shown). Blanks without NADPH or with heat-inactivated protein formed similar amount of metabolites, however in the absence of ascorbic acid, they were not formed.

Table 2. Effect of E2 on ACI hepatic NADPH-dependent Oxidation of E21

Eg Metabolites formed (pMAng/min)_ E:

Table 2. Effect of E2 on ACI hepatic NADPH-dependent Oxidation of E21

Eg Metabolites formed (pMAng/min)_ E:

Treatment

4-OH-E2

2-OH-E2

2-OH-E,

E|

Metabolized (pM/mg/min)

6 Weeks

Chot, 20 mg

9± 2

123 ± 11

24 ±7

134 ± 13

392 ±23 (17%)

E2, 3 mg + Choi, 17 mg

11 ±2

149 ± 10

32 ±6

232 ±12

530 ±28 (25%)

12 Weeks

Choi, 20 mg

7 ± 1

t69± 12

41 ±7

140 ±28

473 ± 33 (24%)

E2, 3 mg + Choi, 17 mg

11 ±3

182 ±36

102 ± 26

309 ±422

644 ± 64 (32%)

1 Data represent the mean ± SE of 6 rats/group.

2 Statistically different from control group (p <0.05),

1 Data represent the mean ± SE of 6 rats/group.

2 Statistically different from control group (p <0.05),

Control liver microsomes incubated with 100 (iM E2 and 2 mM UDPGA resulted in the formation of two metabolites: E2-3p-glucuronide (128 ± 9 pmol/mg/min) and E2-17P-glucuronide (87 ± 13 pmol/mg/min) respectively. The formation ofthese metabolites was not altered by chronic E2 treatment at any of the time periods examined. MG microsomal formation of E2 glucuronides was not detected under the same conditions used for liver microsome.

Liver or MG microsome incubations with in the presence of oleoyl-coenzyme A as a cofactor, followed by HPLC detection, revealed a single radioactive peak less polar than E2 corresponding to E2-17p-oleoyl ester as reported previously (25). Chronic E2 treatment did not alter hepatic esterification, but was reduced by ~ 80 % of control values in the MG (Figure 3). This decrease in MG E2 esterification occurred as early as 6 w. The Vmax but not the Km (8 pM) for this reaction was decreased markedly after E2 treatment (from 6.017 to 2.725 pmol/mg/min).

Figure 3. Effect of chronic E2 treatment on ACO:E2 activity in ACI rat MG and liver. Data represent the mean ± S.E. of 3 rats/group. Data from the control (C) group at different time periods were similar and pooled (n = 9). * Statistically different from C group, p < 0.05.
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