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Toxicology Letters 218 (2013) 18– 23

Contents lists available at SciVerse ScienceDirect

Toxicology Letters

jou rn al h om epa ge: www.elsev ier .com/ locate / tox le t

ffects of methoxychlor and its metabolite 2,2-bis(p-hydroxyphenyl)-1,1,1-richloroethane on 11�-hydroxysteroid dehydrogenase activities in vitro

ingjing Guoa, Haiyun Denga, Hongzhi Lib, Qiqi Zhua, Binghai Zhaob, Bingbing Chenc,anhui Chub, Ren-Shan Gea,∗

The 2nd Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang 325000, PR ChinaHeilongjiang Key Laboratory of Anti-Fibrosis Biotherapy, Mudanjiang Medical University, Heilongjiang, PR ChinaDepartment of Pharmacology of School of Pharmacy, Wenzhou Medical College, Wenzhou, Zhejiang 325000, PR China

i g h l i g h t s

Methoxychlor and HPTE competitively inhibit human 11�-hydroxysteroid dehydrogenase 1.HPTE is more potent than methoxychlor in rats to inhibit 11�-HSD1.HPTE is more potent than methoxychlor in humans and rats to inhibit 11�-HSD2.

r t i c l e i n f o

rticle history:eceived 5 December 2012eceived in revised form 10 January 2013ccepted 10 January 2013vailable online 17 January 2013

a b s t r a c t

Methoxychlor (MXC) is primarily used as a pesticide and widely present in the environment. The objectiveof the present study is to investigate the direct effects of MXC and its metabolite 2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) on two isoforms of 11�-hydroxysteroid dehydrogenase (11�-HSD1 and11�-HSD2) in vitro. Human liver microsome, rat testis microsome and adult Leydig cells were usedfor the measurement of 11�-HSD1 activity. Human placental and rat kidney microsomes were used

eywords:ethoxychlorPTEnzyme inhibition1�-hydroxysteroid dehydrogenase

for 11�-HSD2 activity. The IC50 values on human 11�-HSD1 by MXC and HPTE were 1.91 ± 0.07 and8.88 ± 0.08 �M, respectively. HPTE inhibited rat 11�-HSD1 with IC50 of 9.15 ± 0.05 �M, while MXC didnot inhibit the enzyme. MXC and HPTE were competitive inhibitors of 11�-HSD1. HPTE also inhibitedhuman and rat 11�-HSD2 with IC50 values of 55.57 ± 0.08 and 12.96 ± 0.11 �M, respectively, while MXCdid not inhibit 11�-HSD2. In summary, our results showed that MXC and its metabolite HPTE inhibited

in a

lucocorticoid metabolism both isoforms of 11�-HSD

. Introduction

Dichlorodiphenyltrichloroethane (DDT) is an organochlorineesticide, which was widely used in agriculture before 1972. It haseen considered as a persistent organic pollutant (POP). Methoxy-hlor (MXC) is structurally similar to DDT and considered to behe alternative insecticide to replace DDT due to its relatively lowoxicity and less persistence in the environment. MXC is primar-ly used as a pesticide, which is sprayedon fruits, crops, vegetables,nd home garden to prevent insects (Gupta et al., 2006; Wauchopet al., 1992). MXC exposure primarily occurs via air, soil and water

hat have been contaminated.

After ingestion, MXC is predominantly metabolizednto mono- and bis-demethylated metabolites, including

∗ Corresponding author. Tel.: +86 571 88879169.E-mail addresses: yanhui chu@sina.com (Y. Chu), r ge@yahoo.com,

enshange@gmail.com (R.-S. Ge).

378-4274/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.toxlet.2013.01.002

species- and chemical structure-dependent manner.© 2013 Elsevier Ireland Ltd. All rights reserved.

2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)-1,1,1-trichloroethaneand 2,2-bis(4-hydroxyphenyl)-1,1,1-trichloroethane (HPTE)(Kapoor et al., 1970). Previous observations indicate that bothMXC and HPTE are endocrine disruptors (Hall et al., 1997). In manycases, HPTE has been found to have greater reproductive toxicityin both sexes than parent compound MXC (Gupta et al., 2007). Inthe female rat, HPTE inhibited follicle-stimulating hormone andcAMP-stimulated steroid production by regulating messenger RNAexpression levels of some steroidogenic enzymes in granulosacells of ovary (Harvey et al., 2009). In the male rat, MXC andHPTE were found to inhibit testosterone production in Leydigcells (Akingbemi et al., 2000). We also found that MXC and HPTEdirectly inhibited testosterone biosynthetic enzyme activities,especially the 3�-hydroxysteroid dehydrogenase.

Previous studies demonstrated that glucocorticoids inhibited

testosterone production and caused cell apoptosis of Leydig cellsvia binding to glucocorticoid receptors in this cell type (Gao et al.,2002; Monder et al., 1994b). One group of hydroxysteroid dehy-drogenases, 11�-hydroxysteroid dehydrogenases (11�-HSDs) are

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ctively involved in the glucocorticoid metabolism. Two isoforms,ype I (11�-HSD1) and type II (11�-HSD2) have been clonedAgarwal et al., 1989, 1994). Both enzymes are located in smoothndoplasmic reticulum (Agarwal et al., 1989; Albiston et al.,994). They possess distinct biochemical characteristics: 11�-SD1 is a low-affinity NADP+/NADPH-dependent bidirectionalnzyme (Monder and White, 1993) and 11�-HSD2 is a high-affinityAD+-dependent unidirectional enzyme (White et al., 1997a). Ley-ig cells contain both isoforms of 11�-HSD (Ge et al., 1997, 2005a).

n the Leydig cell, 11�-HSD1 is primarily an oxidase and 11�-SD2 is a unidirectional oxidase (Ge et al., 1997, 2005a). Thus, bothnzymes in the Leydig cell prevent glucocorticoid-induced inhi-ition of testosterone production by oxidatively inactivating thective glucocorticoid (Ge et al., 1997, 2005a). However, the directnhibitory actions on both 11�-HSD enzyme activities by MXC andPTE have not been investigated. The objective of the present studyas to study the direct effects of MXC and HPTE on both isoforms

f 11�-HSD and the mode of inhibition in vitro.

. Materials and methods

.1. Materials

[1,2,6,7-3H] Corticosterone (3H-CORT) and [1,2,6,7-3H] cortisol (3H-cortisol)ere purchased from Dupont-New England Nuclear (Boston, MA). 3H-11-ehydrocorticosterone (3H-11DHC) and 3H-cortisone were prepared from labelledH-CORT or 3H-cortisol as described earlier (Lakshmi and Monder, 1985). Cold CORT,1DHC, cortisol and cortisone were purchased from Steraloids (Newport, RI). MXC99.5% purity) was purchased from Sigma (St. Louis, MO). HPTE was a gift from Dr

.R. Kelce (Monsanto Company, St Louis, MO, USA). MXC was dissolved in dimethylulfoxide (DMSO). Sprague Dawley rats were purchased from Laboratory Animalentre of Wenzhou Medical College. The Institutional Animal Care and Use Commit-ee of Wenzhou Medical College approved all procedures. Human liver and placental

icrosomes were purchased from Hangzhou Yihaixuan Biotech (Hangzhou, China),hich obtained from a pool of healthy donors. The use of human microsomes was

ranted by our institution (Wenzhou Medical College Research Ethics Committee).

.2. Preparation of microsomal protein

Male adult rat testis that abundantly contains 11�-HSD1 was used for the prepa-ation of microsomes to measure 11�-HSD1 activity (Ge et al., 1997). Male adultat kidney that abundantly contains 11�-HSD2 was used for the preparation oficrosomes to measure 11�-HSD2 activity (Moore et al., 2000). The preparation

f microsomes was performed as described in our previous study (Ge et al., 1997).n brief, rat testis or kidney was homogenized in 0.01 mM PBS buffer containing.25 M sucrose, and nuclei and large cell debris were removed by centrifugationt 1500 × g for 10 min. The post-nuclear supernatants were centrifuged twice at05,000 × g, the resultant microsomal pellets were re-suspended. Protein contentsere measured by Bio-Rad Dye Reagent Concentrate (Cat.# 500-0006). The concen-

rations of rat testis or kidney microsomes were 20 mg/ml. Microsomes were usedor measurement of 11�-HSD1 and 11�-HSD2 activities.

.3. Leydig cell isolation

Purified rat Leydig cells were obtained from 90-day-old Sprague Dawley ratsy collagenase digestion of the testes followed by Percoll density centrifugation ofhe cell suspension, according to the previously described method (Sriraman et al.,001). Adult Leydig cells were harvested from the Percoll gradient at a band at.070 mg/ml. The purity of cell fractions was evaluated by histochemical stainingor 3�-hydroxysteroid dehydrogenase activity with 0.4 mm etiocholanolone as theteroid substrate (Payne et al., 1980). Enrichment of rat Leydig cells was typicallyore than 95%.

.4. 11ˇ-HSD1 assay in rat testis and human liver microsomes

11�-HSD1 activity assay tubes contained 25 nM substrate 11DHC (for rat) or cor-isone (for human), spiked with 30,000 cpm of their respective 3H-steriods. 25 nMf steroid substrate was used because this concentration was within the physiolog-cal concentration range (Blanchard et al., 1995). The rat testis microsomes (10 �g)r human liver (4 �g) microsomes were incubated with substrate, 0.2 mM NADPHnd various concentrations of MXC or HPTE at 37 ◦C for 60–90 min. The inhibitory

otency of MXC or HPTE was measured relative to control (only DMSO). MXC orPTE was dissolved in DMSO with final concentration of 0.4%, at which concentra-

ion DMSO did not inhibit this enzyme activity. The reaction was stopped by adding0 �l of 1 mM glycyrrhentic acid and 1 ml ice-cold ether. The steroids were extracted,nd the organic layer was dried under nitrogen. The steroids were separated

ers 218 (2013) 18– 23 19

chromatographically on the thin layer plate in chloroform and methanol (90:10,v/v), and the radioactivity was measured using a scanning radiometer (SystemAR2000, Bioscan Inc., Washington, DC) as described previously (Ge et al., 1997).The percentage conversion of 11DHC to CORT or cortisone to cortisol was calcu-lated by dividing the radioactive counts identified as 11-OH-steroids by the totalcounts.

2.5. 11ˇ-HSD1 assay in the intact adult Leydig cells

Because intact rat Leydig cells can maintain both 11�-HSD1 oxidase and reduc-tase activity, its oxidation or reduction was also measured in intact Leydig cells usingendogenous NADP+ or NADPH (Ge et al., 1997), the 11�-HSD1 oxidase assay tubescontained 25 nM CORT spiked with 30,000 cpm of [3H]CORT and 0.025 × 106 cells,and the mixture was incubated for 30 min. The 11�-HSD1 reductase assay tubescontained 25 nM 11DHC spiked with 30,000 cpm of [3H]11DHC and 0.045 × 106

cells, and the mixture was incubated for 120 min. The rest procedure was asdescribed above. The 11�-HSD1 assays in intact Leydig cells were repeated by fourtimes.

2.6. 11ˇ-HSD2 assay in rat kidney microsomes

11�-HSD2 activity assay tubes contained 25 nM CORT (within the range of phys-iological levels). [3H] CORT was used as the substrate to measure rat 11�-HSD2activity. Kidney microsome (10 �g) was incubated with substrate and 0.2 mM NAD+

for 30 min. The rest procedure was as described above. The percentage conversionof CORT to 11DHC was calculated by dividing the radioactive counts identified as11DHC by the total counts associated with both substrate and product.

2.7. 11ˇ-HSD2 assay in human placental microsomes

Because human placenta abundantly contains 11�-HSD2 enzyme (Hirasawaet al., 2000), human placenta was used as enzyme source for measurement of11�-HSD2 activity. Assay tubes contained 25 nM cortisol (within the range of phys-iological levels). [3H] cortisol was used as a substrate to measure human 11�-HSD2activity. Placental microsome (6 �g) was incubated with cortisol and 0.2 mM NAD+

for 30 min as described in Section 2.6. The rest procedure was as described above.The percentage conversion of cortisol to cortisone was calculated by dividing theradioactive counts identified as cortisone by the total counts associated with bothsubstrate and product.

2.8. Determination of half maximum inhibitory concentrations (IC50) andinhibitory mode

The IC50 was determined by adding 25 nM substrate with 0.2 mM cofactor andvarious concentrations of MXC or HPTE at 250 �l reaction buffer (0.1 mM PBS)containing rat or human microsomal proteins as described previously (Guo et al.,2012).In order to measure the IC50 values of 11�-HSD1 activity, we added 10 �grat testis microsomes or 4 �g human liver microsomes into the reaction buffer andincubated the reaction mixture for 60–90 min. For measuring the IC50 values of 11�-HSD2 activity, we added 10 �g rat kidney microsomes or 6 �g human placentalmicrosomes into the reaction buffer and incubated the reaction mixture for 60 min.The method for the mode of inhibition was similar to the measurement of IC50 exceptfor adding various concentrations of substrate or cofactor. Both MXC and HPTE wereassayed at the same conditions.

2.9. Statistics

Each experiment was repeated four times. Data were subjected to nonlinearregression analysis by GraphPad (Version 5, GraphPad Software Inc., San Diego, CA)for IC50 values. Lineweaver–Burk plot was used for the analysis of the mode of action.Data were subjected to analysis by ANOVA followed by ad hoc Tukey comparisonto identify significant differences between control (CON) and MXC or HPTE groups.All data are expressed as means ± SEM. Differences were regarded as significant atP < 0.05.

3. Results

3.1. The effects of MXC and HPTE on 11ˇ-HSD1 activity

The conversion of cortisone to cortisol in human liver micro-somes or 11DHC to CORT in rat testis microsomes has been shownto be catalyzed in a NADPH-dependent manner by human orrat 11�-HSD1 reductive activity in the microsomes. As shown in

Table 1, the human liver and rat testis 11�-HSD1 activities were7.26 ± 0.32 pmol cortisol/mg protein.min and 4.96 ± 0.01 pmolCORT/mg protein.min, respectively. MXC and HPTE significantlyinhibited human 11�-HSD1 reductase. MXC was more potent than

20 J. Guo et al. / Toxicology Letters 218 (2013) 18– 23

Table 1The inhibitory potencies of MXC and HPTE on 11�-HSD1 and 11�-HSD2.

11�-HSD1 11�-HSD2Human Rat Human Rat

Activity (pmol/mg.min) 7.26 ± 0.32 4.96 ± 0.01 3.59 ± 0.39 6.99 ± 0.24IC50 (�M) for 11�-HSD1 IC50 (�M) for 11�-HSD2Human Rat Human Rat

MXC 1.91 ± 0.07 NI NI NI9.15 ± 0.05 55.57 ± 0.08 12.96 ± 0.11

N

HaHMmtMfaHdi

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Fig. 2. Effects of MXC on rat 11�-hydroxysteroid dehydrogenase 1 (11�-HSD1)activities. 11�-HSD1reductase in rat testicular microsome (black bar), intact rat

HPTE 8.88 ± 0.08

I = no inhibition at 100 �M. Mean ± SEM, n = 4.

PTE to inhibit human 11�-HSD1, with IC50 values of 1.91 ± 0.07nd 8.88 ± 0.08 �M, respectively (Fig. 1 and Table 1). In contrast,PTE was more potent to inhibit rat 11�-HSD1 reductase thanXC, with the Panel B, IC50 value of 9.15 ± 0.05 mM, while MXCinimally inhibited rat microsomal activity at higher concentra-

ion (Fig. 1, Table 1). When intact rat Leydig cells were used,XC showed much higher potency compared to the data obtained

rom rat testicular microsome. MXC inhibited 11�-HSD1 reductivectivity in intact rat Leydig cells by about 93% (Fig. 2) at 100 �M.owever this concentration of MXC did not inhibit 11�-HSD1 oxi-ase activities at all, suggesting that 11�-HSD1 reductase activity

s more sensitive to MXC inhibition in intact Leydig cells.Both MXC (Fig. 3A) and HPTE (Fig. 3B) competitively inhibited

uman 11�-HSD1 reductase when steroid substrates were used.PTE also competitively inhibited rat 11�-HSD1 reductase against

ubstrate CORT (Fig. 4A), and exerted uncompetitive inhibition onat enzyme against cofactor NADPH (Fig. 4B).

ig. 1. Concentration-dependent effects of MXC and HPTE on human and rat 11�-ydroxysteroid dehydrogenase 1 (11�-HSD1) activities for 60–90 min incubation.he conversion rates of 11�-HSD1 in human liver and rat testis microsomes in thesexperiments were 40–50%. Values from four samples are represented.

Leydig cells (white bar) and 11�-HSD1oxidase (stripped bar) in intact rat Leydigcells were measured. Mean ± SEM, n = 4. Identical letters designate no significantdifferences between two groups at P < 0.05.

3.2. The effects of MXC and HPTE on 11ˇ-HSD2 activity

The conversion of cortisol to cortisone in human placentalmicrosomes or CORT to 11DHC in rat kidney microsomes has beenshown to be catalyzed in a NAD+-dependent manner by human or

rat 11�-HSD2 in the placental or kidney microsomes. When thehighest concentration (100 �M) was tested, MXC did not inhibitboth human and rat 11�-HSD2 activities, while HPTE significantly

Fig. 3. The inhibitory modes of MXC and HPTE on human 11�-hydroxysteroid dehy-drogenase 1. Lineweaver–Burk plots in presence of cortisone and MXC (Panel A) andcortisone and HPTE (Panel B). Values were obtained from four samples.

J. Guo et al. / Toxicology Letters 218 (2013) 18– 23 21

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Fig. 5. Effects of MXC and HPTE on human and rat 11�-hydroxysteroid dehydroge-nase 2 (11�-HSD2) activities. 11�-HSD2 activities were measured in the presence

contrast, its metabolite HPTE inhibited this enzyme in both humanand rat microsomes.

11�-HSD1 plays many physiological roles. In the testis, 11�-HSD1 exclusively resides in Leydig cells (Phillips et al., 1989), and

ig. 4. The inhibitory mode of HPTE on rat 11�-hydroxysteroid dehydrogenase 1.ineweaver–Burk plots in presence of 11DHC and HPTE (Panel A) and NADPH andPTE (Panel B). Values were obtained from four samples.

nhibited both enzyme activities (Fig. 5a). HPTE inhibited rat 11�-SD2 activity more potently than human enzyme, with IC50 valuesf 12.96 ± 0.11 and 55.57 ± 0.08 �M, respectively (Fig. 6). HPTEompetitively inhibited human (Fig. 7A) and rat (Fig. 7B) 11�-HSD2ctivities, when steroid substrates were used. HPTE exerted uncom-etitive inhibition on both human and rat 11�-HSD2 activitiesgainst cofactor NAD+ (data not shown).

. Discussion

Many DDT isomers, including MXC, continue to be used for agri-ultural purposes and are present in the environment. Thus, theseompounds pose a health risk not only to agricultural workers butlso to the general population. The results presented herein identifydditional mechanisms of MXC and HPTE action regulating glu-ocorticoid metabolism and reinforce previous observations thathese compounds have the capacity to cause adverse biologicalffects affecting the functions of testis and other tissues. Identi-cation of the mechanisms of action of hormonally active agentsresent in the environment is required to facilitate the process ofisk assessment of the population.

It is true that MXC and HPTE also have different potencies ofnhibiting several other human hydroxysteroid dehydrogenases.PRE inhibited human testicular 3�-HSD with IC50 of 8.3 �M, whileXC had IC50 of 53.2 �M (Hu et al., 2011). HPTE inhibited human

7�-HSD3 with IC50 of 12.1 �M, while MXC did not affect thisnzyme activity at concentrations as high as 100 �M (Hu et al.,011). Unlike the inhibition of 3�-HSD and 17�-HSD3 by MXCetabolite HPTE, MXC is not required to be metabolically acti-

ated in the human tissues to inhibit 11�-HSD1. In contrast, MXC isequired to be activated into HPTE in the rat tissues to inhibit both

1�-HSD isoforms.

11�-HSDs are critical enzymes to regulate local concentrationsf glucocorticoid, thus controlling both glucocorticoid and miner-locorticoid actions. Two isoforms, 11�-HSD1 and 11�-HSD2, are

of 25 nM cortisol (human, Panel A) and corticosterone (rat, Panel B) and 100 �MMXC or HPTE. Mean ± SEM, n = 6. Identical letters designate no significant differencesbetween two groups at P < 0.05.

differently expressed in tissues in the distinct patterns to modulatethese processes. In the present study, we observed dose-dependentinhibition of human and rat 11�-HSD1 by MXC and its metaboliteHPTE. MXC is more potent than HPTE to inhibit human 11�-HSD1,while HPTE is more potent than MXC to inhibit rat 11�-HSD1 activ-ity. MXC did not inhibit both human and rat 11�-HSD2 activities. In

Fig. 6. Concentration-dependent inhibitions of HPTE on human and rat 11�-hydroxysteroid dehydrogenase 2 (11�-HSD2) activities. Values from four samplesare represented.

22 J. Guo et al. / Toxicology Let

Fig. 7. The inhibitory modes of HPTE on human and rat 11�-hydroxysteroid dehy-da

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rogenase 2. Lineweaver–Burk plots in presence of cortisone and HPTE (Panel A)nd corticosterone and HPTE (Panel B). Values were obtained from four samples.

ts activity in Leydig cells is much higher than that of liver cellser se (Monder et al., 1994a). 11�-HSD2 is also expressed in Ley-ig cells, although its amount is much lower than 11�-HSD1 (Get al., 2005b). However, this high affinity unidirectional oxidaseompletely converts active glucocorticoid to inactive one (Whitet al., 1997a). 11�-HSD1 is a bidirectional oxidoreductase (Ge et al.,997). However, in Leydig cells, 11�-HSD1 is a primary oxidase (Get al., 1997). Both 11�-HSD1 and 11�-HSD2 have been suggestedo protect this cell type from glucocorticoid-mediated inhibitionf testosterone production by inactivating the active glucocorti-oid (Ge et al., 1997, 2005b). Thus, the inhibition of 11�-HSD1 and1�-HSD2 activities in Leydig cells could alter testosterone pro-uction.

11�-HSD1 is also abundantly present in human and rodentiver and acts as a predominant reductase which generates activelucocorticoid locally (Agarwal et al., 1995; Ge et al., 1997). Thelucocorticoid has been found to profoundly regulate the glu-ose metabolism in the liver (Yabaluri and Bashyam, 2010). Thenhibition of 11�-HSD1 by MXC and HPTE may therefore disruptegulation of glucose metabolism.

11�-HSD2 is primarily expressed in the human and rat kidney, mineralocorticoid receptor target tissue (Agarwal et al., 1994;tewart et al., 1994). The endogenous ligand for mineralocorticoideceptor is aldosterone. Aldosterone binds to mineralocorticoideceptor to induce the transcription of its target genes, thus increas-ng sodium and water retention. The mineralocorticoid receptor hashe similar affinities for aldosterone and cortisol and cannot distin-uish these two ligands (Arriza et al., 1987). Therefore, 11�-HSD2cts as a gate keeping protein by eliminating active glucocorti-oid (White et al., 1997a). The null mutation of 11�-HSD2 gene or

he inhibition of this enzyme by chemicals caused accumulation ofctive glucocorticoid in the kidney, leading to the apparent miner-locorticoid excess syndrome, in which patients have hypertension

ters 218 (2013) 18– 23

and hypokalemia (Funder et al., 1988; Stewart et al., 1987; Whiteet al., 1997b).

11�-HSD2 is also localized in the placenta, a glucocorticoidtarget tissue. 11�-HSD2 is expressed in the syncytiotrophoblast,which is responsible for maternal-foetal nutrition exchange(Krozowski et al., 1995). Placental 11�-HSD2 is critical for protec-ting the foetus from overexposure to maternal cortisol, which willblock foetal development (Doyle et al., 2003; Seckl, 2004; Seckland Holmes, 2007). Therefore, the placental 11�-HSD2 plays animportant role in maintaining pregnancy and promoting the foetalmaturation.

The present study showed that MXC and HPTE had differentpotencies of inhibiting human or rat 11�-HSD1 and 11�-HSD2.MXC was more potent to inhibit human 11�-HSD1 than HPTE. Incontrast, HPTE inhibited rat 11�-HSD1 more potently than MXC.MXC and HPTE also had different potencies of inhibiting human andrat 11�-HSD2. At 100 �M, MXC did not inhibit human or rat, whileHPTE potently inhibited both human and rat 11�-HSD2. Causes ofthese differences are unclear. We speculate that the human and rat11�-HSD1 or 11�-HSD2 has different affinity for MXC or HPTE.).

The human exposure of MXC is varied. The serum MXC levelsin 15 human subjects in the Honduras were from 0.24 to 4.07 mg/L(Steinberg et al., 1989). This concentration is within the range ofconcentrations, at which MXC inhibited human 11�-HSD1 activity.

In conclusion, MXC or HPTE is an inhibitor of 11�-HSD1 and2 enzyme activities. However, the relation of these results to theeffects in the whole animal, a future study of measuring in vivo 11�-HSD1 and 11�-HSD2 activities is required to address its usefulnessto risk assessment.

Acknowledgement

This research was partially supported by NSFC (81102092 to B.B.C, 81070329 to Y.H. Chu, 31171425 to R.S.G).

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