DOSE–RESPONSE AND TIME COURSE RELATIONSHIPS FOR VITELLOGENIN INDUCTION IN MALE WESTERN FENCE LIZARDS (SCELOPORUS OCCIDENTALIS) EXPOSED TO ETHINYLESTRADIOL

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  DOSE–RESPONSE AND TIME COURSE RELATIONSHIPS FOR VITELLOGENIN INDUCTION IN MALE WESTERN FENCE LIZARDS (SCELOPORUS OCCIDENTALIS) EXPOSED TO ETHINYLESTRADIOL
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  1410 Environmental Toxicology and Chemistry, Vol. 21, No. 7, pp. 1410–1416, 2002   2002 SETACPrinted in the USA0730-7268/02 $9.00    .00 DOSE–RESPONSE AND TIME COURSE RELATIONSHIPS FOR VITELLOGENININDUCTION IN MALE WESTERN FENCE LIZARDS ( SCELOPORUS OCCIDENTALIS  )EXPOSED TO ETHINYLESTRADIOL S ANDRA  M. B RASFIELD , L YNN  P. W EBER , L ARRY  G. T ALENT , and D AVID  M. J ANZ * Department of Zoology, Oklahoma State University, Stillwater, Oklahoma 74078, USA(  Received   8  August   2001;  Accepted   27  November   2001) Abstract —The long-term goal of this research is to develop and validate an in vivo reptile model for endocrine-mediated toxicityusing fence lizards ( Sceloporus  spp.). One of the best defined estrogenic responses in oviparous vertebrates is induction of the yolk precursor protein, vitellogenin (Vtg). In this study, dose–response and time course relationships for Vtg induction were determinedin male western fence lizards ( Sceloporus occidentalis ) given intraperitoneal injections of 17  -ethinylestradiol (EE 2 ). Plasma Vtgwas quantified directly with an antibody-capture enzyme-linked immunosorbent assay (ELISA) and indirectly using plasmaalkaline-labile phosphate (ALP) in order to compare these two methods. Both ELISA and ALP predicted similar median effective dose(ED50 [dose causing a 50% maximal response]) values for plasma Vtg induction (0.167 mg/kg for ELISA and 0.095 mg/kg forALP). In addition, both ELISA and ALP detected significant Vtg induction at a dose of 0.0003 mg/kg of EE 2 , which was the lowestdose used in our study. A decrease in body weight at the highest dose (10 mg/kg) and an increase in hepatosomatic index at thefour highest doses were observed. Serial dilutions of plasma from an EE 2 -exposed male revealed a high correlation between plasmaVtg and ALP determinations in this species. In conclusion, our data show that plasma ALP may be a suitable alternative formeasuring plasma Vtg compared with developing a Vtg ELISA in fence lizards exposed to estrogenic compounds. Keywords —Vitellogenin Alkaline-labile phosphate Reptiles Estrogenic Ethinylestradiol INTRODUCTION In recent years, it has become increasingly clear that chem-icals in the environment from both natural and anthropogenicsources may interfere with endocrine physiology [1–3]. How-ever, data are often insufficient to resolve the ecological risk associated with endocrine-disrupting chemicals (EDCs) thatexert toxicity through alterations in endocrine systems. Suit-able test systems and appropriate endpoints are generally notavailable to assess the significance of the exposure and impactsof many chemicals in the environment [4]. There is a recog-nized need to develop animal models for all vertebrate classes[1,5]. In particular, existing methods suitable for identifyingchemicals with endocrine-disrupting mechanisms in reptilesare limited [1,5,6]. We hypothesize that fence lizards ( Scelo- porus  spp.) are good candidates for laboratory reptile modelsfor assessing endocrine-mediated toxicity in vivo.Reptiles are considered to be suitable as contaminant biom-onitors due to their persistence in a variety of habitats, widegeographic distribution, longevity, and site fidelity [3]. How-ever, with the exception of a few species, this class of verte-brates has been relatively understudied [6]. Currently, the red-eared slider turtle ( Trachemys scripta elegans ) [7] and Amer-ican alligator (  Alligator mississippiensis ) [3,8] are being eval-uated for use in assessment of endocrine-mediated toxicity.In comparison with other reptile models, fence lizards mayrepresent ideal candidates due to their wide geographic dis-tribution, small size, high fecundity, short time to sexual ma-turity, and early development of secondary sexual character-istics. Together, eastern ( Sceloporus undulatus ) and western( Sceloporus occidentalis ) fence lizards inhabit most of the * To whom correspondence may be addressed(djanz@okstate.edu). continental United States and are common throughout thisrange [9]. Although the natural history characteristics of east-ern and western fence lizard populations may vary under nat-ural conditions, many populations mature under laboratoryconditions in as few as four to eight months after hatching[10], and mature females typically lay 2 to 6 clutches of be-tween 8 and 15 eggs in a breeding season [11]. Individualsare easily sexed after hatching and offer a variety of endpointsthat can be evaluated relative to endocrine-mediated toxicity.The induction of Vtg in male oviparous vertebrates rep-resents a sensitive biochemical endpoint indicating exposureto estrogenic compounds [1,12]. Vitellogenin is a high mo-lecular weight phosphoglycolipoprotein produced in the liverof all oviparous vertebrates in response to circulating levelsof the endogenous estrogen 17  -estradiol [13]. In females, Vtgis then secreted into systemic circulation for transport to theovary, where it is actively incorporated into developing oo-cytes. Under normal physiological conditions, Vtg is a female-specific protein since males have very low circulating levelsof endogenous estradiol. However, males do have the capacityto express this protein if exposed to exogenous estrogens.Thus, the presence of Vtg in the plasma of male oviparousvertebrates indicates a physiological response to exogenousestrogens [1,12]. This is an ideal endpoint to measure in malesbecause the majority of Vtg remains in systemic circulationrather than being sequestered into oocytes, as would occur infemales [14].Vitellogenin is a highly phosphorylated protein [13], anda measure of the phosphate associated with this protein hasbeen used as an indicator of plasma Vtg concentration [15–18]. Plasma phosphoproteins can be separated fromotherphos-phorylated plasma molecules (e.g., phospholipids) with a se-ries of extractions. Because Vtg is the major plasma phos-  Biochemical responses of fence lizards to estrogen exposure  Environ. Toxicol. Chem.  21, 2002 1411 phoprotein in an estrogen-exposed oviparous animal [15], sim-ple colorimetric measurement of alkaline-labile phosphate(ALP) released from extracted plasma phosphoproteins hasbeen used as an indirect measure of circulating Vtg in fishes[16–19]. In rainbow trout ( Oncorhynchus mykiss ), increasinglevels of plasma Vtg were correlated with increases in plasmaALP [19]. However, the relationship between plasma vitello-genin and ALP has not been characterized in reptiles.As an initial step toward evaluating fence lizards as a lab-oratory reptile model for endocrine-mediated toxicity, the pre-sent study determined dose–response and time course rela-tionships of Vtg induction in males exposed to EE 2 . A mainobjective of this study was to compare two methods of mea-suring this response, a direct measure of Vtg using an enzyme-linked immunosorbent assay (ELISA) and an indirect measureof the ALP associated with this protein. MATERIALS AND METHODS  Animals Male western fence lizards ( S. occidentalis ) from Reno(NV, USA) and San Joaquin Valley (CA, USA) were accli-mated to laboratory conditions prior to use. Animals werehoused in glass aquaria with a constant photoperiod of 14:10h light:dark. Food (crickets) and water were provided ad li-bitum. Chemicals 17  -Ethinylestradiol (EE 2 , purity 98%) and all other chem-icals were obtained from Sigma Chemical (St. Louis, MO,USA) unless otherwise specified. Preliminary dose–response and time course experiments In preliminary experiments, we examined the effects of single and multiple intraperitoneal (i.p.) injections of EE 2  inmale fence lizards ( n    4 lizards) and analyzed plasma Vtgboth directly by ELISA and indirectly using plasma ALP. TheEE 2  was dissolved in acetone and added to a carrier solutionof corn oil. The acetone was then evaporated under a streamof nitrogen. All experimental groups, including vehicle con-trols, received an i.p. injection of 5   l stock solution/g bodyweight. Two injection regimens were compared, a single bolusinjection and five additive doses administered every secondday. Doses included corn oil vehicle controls and 0.001, 0.01,0.1, 1, and 10 mg EE 2  /kg body weight. An additional exper-iment was also conducted to examine the effect of vehicles(corn oil and propylene glycol) on Vtg induction. Animalswere given a single injection of vehicle at a volume of 5   lstock solution/g body weight or sham injection.Blood samples were repeatedly collected on days 3, 6, 9,15, 21, and 27 in the single-dose groups and on days 9, 12,15, 21, and 27 in the multiple-injection groups from the samelizards. Blood (  100   l) was collected from the postorbitalsinus using a heparinized microcapillary tube and placed in atube containing 5  l of 5.12 mg/ml aprotinin and 5  l of 1.36mg/ml heparin in normal saline to inhibit proteolysis and clot-ting, respectively. Plasma was collected following centrifu-gation at 5,000 rpm for 10 min at 4  C. Samples were storedat  80  C until analysis. Animals were killed on day 27, bodyweights were recorded, and livers were excised and weighed.  Dose–response experiment  Single i.p. injections of a wider range of EE 2  doses wereadministered to male lizards from Reno in order to describethe dose–response curve for Vtg induction and generate ED50values. Stock solutions of EE 2  were prepared as describedabove using propylene glycol as a carrier. Based on data fromthe preliminary experiments, the experimental groups ( n    8lizards per dose) chosen were sham control, propylene glycolvehicle control, and 0.0003, 0.003, 0.001, 0.01, 0.1, 1, and 10mg/kg. A single i.p. injection (5   l/g, except sham) was ad-ministered and lizards were killed after blood was collectedon day 15. Body weights were recorded before treatment onday 0 and before blood collection on day 15. Corrected he-patosomatic indices were calculated using the formula (liverwt/[body wt    liver wt])    100. Comparison of EE  2  and 17    -estradiol (E  2 ) Stock solutions of EE 2  and E 2  were prepared as describedabove using propylene glycol as a carrier. Compounds wereadministered at a dose of 0.167 mg/kg to compare the differ-ential induction of Vtg. Lizards from San Joaquin Valley ( n   8 per group) received an i.p. dose at an injection volumeof 5  l/g body weight. Blood was collected at day 15 andplasma was stored at   80  C until time of analysis. Vitellogenin ELISA Vitellogenin was isolated from plasma collected from malefence lizards injected i.p. with EE 2  by precipitation and pu-rified using diethylaminoethyl Sephacel protein chromatog-raphy [20]. Highly specific polyclonal antisera against purifiedfence lizard Vtg were produced in rabbits (L.P. Weber et al.,unpublished data). Microtiter plates (Greiner America, LakeMary, FL, USA) were coated with purified Vtg (200 ng/well)using sodium carbonate buffer (0.1 M NaCO 3 , pH 9.6) over-night at 4  C. Anti-Vtg antiserum (1:200) was preincubatedwith standards (2.5–100,000 ng Vtg/ml) or diluted samples (1:10 for control and at least 1:160 for treated) in phosphate-buffered saline (pH 7.4) containing 0.05% Tween 20 (PBS-T)on a rotating platform for 14 to 16 h at 4  C. Plates were washedthree times with PBS-T between each ELISA step. Coatedplates were blocked with 1% bovine serum albumin in PBS-T for 1 h at room temperature. Preincubated standards wereadded to coated, blocked plates in duplicate and samples wereadded in triplicate, then incubated for 2 h at room temperature.Alkaline phosphatase-conjugated goat antirabbit secondaryan-tibody (1:1,000 in PBS-T) was added to plates, incubated atroom temperature for 1 h, and detected with  p -nitrophenylphosphate solution (0.91 mg/ml in 10% [v/v] diethanolaminebuffer, pH 9.6). Color was allowed to develop in the dark atroom temperature for 40 min (B o    0.9–1.0) and absorbancevalues measured at 405 nm using a microplate spectrophotom-eter (Molecular Devices, Sunnyvale, CA, USA). Nonspecificbinding was determined in uncoated wells without primaryantibody, and maximum binding of anti-Vtg (B o  or zero stan-dard) was determined in coated wells in every assay.Intraassay coefficient of variation for the ELISA was 4.9%( n    6 determinations). Interassay variability was 13.3% ( n  12). The detection limit of the Vtg ELISA was 0.25 ng/ml.Parallelism was observed between the standard curve and se-rially diluted plasma samples. Internal controls were run induplicate on every plate and assays were repeated if valuesdeviated   10% from previously determined values. Plasma alkaline-labile phosphate Using the same plasma samples assayed in the ELISA,extractions for ALP were performed as described by Wallace  1412  Environ. Toxicol. Chem.  21, 2002 S.M. Brasfield et al.Fig. 1. Time course and preliminary dose–response relationships of plasma vitellogenin (Vtg) induction in male western fence lizards ( n   4 per dose) treated with ethinylestradiol, either as a single intra-peritoneal (i.p.) injection ( A ) or five additive i.p. doses administeredevery second day ( B ).Fig. 2. Effect of corn oil and propylene glycol (PPG) vehicles (intra-peritoneal injection) on plasma vitellogenin (Vtg) induction in malewestern fence lizards ( n    4) compared with sham injection. Signif-icant increases in Vtg induction were seen with administration of cornoil compared with PPG (  p    0.03 using repeated measures two-wayanalysis of variance [ANOVA] across all time points). and Jared [15] with minor modifications. Briefly, 5  l of plas-ma and 30  l of 1% bovine serum albumin were added to 1.5ml of 10% (w/v) trichloroacetic acid (TCA), then allowed toprecipitate overnight at 4  C. Pellets were repeatedly washedand centrifuged at 7,000 rpm for 10 min with ice-cold 5% (w/ v) TCA (30 min at 50  C), 100% ethanol (80  C for 1 min),chloroform:ether:ethanol (1:2:2), acetone, andether.Followingthe ether wash, pellets were allowed to dry and then werereconstituted in 250  l of 2 N sodium hydroxide and incubatedfor 15 min at 100  C. The samples were then neutralized byadding 250   l of 2 N HCl. Extracts were stored at   20  C.Extracts were diluted (1:10 for controls and at least 1:100 fortreated) using a 1:1 mixture of 2 N sodium hydroxide and 2N HCl, were assayed in duplicate, and inorganic phosphatewas determined by colorimetric assay using a commerciallyavailable kit (Sigma 670). To correct for bovine serum albuminadded prior to extraction, 30  l of 1% bovine serum albuminwas extracted, determined for every assay, and all sampleswere corrected based on this determination.Intraassay coefficient of variation for plasma ALPwas5.6%( n    6). Interassay variability was 10.7% ( n    12). The de-tection limit of the plasma ALP assay was 0.006   M phos-phate. Parallelism was observed between the standard curveand serially diluted plasma samples.  Data analysis Time course data from the preliminary experiments wereanalyzed using two-way repeated measures analysis of vari-ance (ANOVA) followed by Fisher’s protected least significantdifference posteriori tests as appropriate. All other data wereanalyzed using Student’s  t   test or one-way ANOVA, followedby Dunnett’s posteriori tests as appropriate. The ED50 valueswere calculated using nonlinear, four-parameter logistic re-gression. A value of   p    0.05 was considered statisticallysignificant. Data are expressed as mean    standard error of the mean (SEM). Sample size ( n ) indicates the number of lizards used. RESULTS In preliminary experiments, plasma Vtg concentrations in-creased dose dependently in male lizards ( n    4) receiving asingle i.p. dose of EE 2  (Fig. 1A). For all doses, plasma Vtgwas increased on day 3 of blood collection and remained el-evated over the 27-d course of the experiment. Experimentalgroups ( n  4 per group) receiving multiple i.p. doses of EE 2 displayed high levels of plasma Vtg on day 9 of blood col-lection (Fig. 1B). Unlike the single injection groups, the levelsof plasma Vtg consistently decreased over the 27-d course of the experiment at the two highest doses.Before initiation of the dose–response experiment, wechose to investigate the effect of injection vehicle on Vtginduction. Corn oil vehicle caused a significantly higher in-duction of Vtg in comparison with propylene glycol (Fig. 2,  p    0.03, repeated measures ANOVA across all time points)with peak Vtg levels observed 21 d after a single injection.Plasma Vtg levels in the sham injection group were not sig-nificantly different from either corn oil (  p  0.07) or propyleneglycol (  p    0.63) vehicle controls (Fig. 2).Based on our preliminary time course experiment, day 15following a single i.p. injection was chosen for the dose–re-sponse experiment because it appeared to be a consistent pointof maximum Vtg induction. In this experiment, plasma Vtgfollowed a sigmoidal dose–response relationship, and fromthis  Biochemical responses of fence lizards to estrogen exposure  Environ. Toxicol. Chem.  21, 2002 1413Fig. 3. Plasma vitellogenin (Vtg) levels in untreated (sham injectionand propylene glycol vehicle [PPG] controls) and ethinylestradiol-treated male western fence lizards determined using enzyme-linkedimmunosorbent assay (ELISA). Plasma samples were collected 15 dafter a single intraperitoneal injection. Values are mean    standarderror of the mean ([SEM] [ n    7–8]). From this curve, a medianeffective dose (ED50) value of 0.167 mg/kg was calculated for Vtginduction.Fig. 5. Time course and preliminary dose–response relationships of plasma alkaline-labile phosphate (ALP) induction in male westernfence lizards ( n    4) treated with ethinylestradiol either as a singleintraperitoneal injection ( A ) or five additive i.p. doses administeredevery second day ( B ).Fig. 4. Effects of ethinylestradiol treatmenton ( A )body weightchange(from day of injection to day 15) and ( B ) corrected hepatosomaticindex as compared with sham injection and propylene glycol (PPG)controls in western fence lizards. Animals received a single intraper-itoneal injection and were killed on day 15. Values are mean  stan-dard error of the mean ([SEM] [ n    7–8]). Letters indicate signifi-cantly different from PPG controls (a,  p    0.05; b,  p    0.01) inDunnett’s a posteriori test after one-way analysis of variance (AN-OVA). curve, an ED50 value of 0.167 mg/kg (95% confidence limit:0.040–0.70 mg/kg) was calculated (Fig. 3). The equation de-scribing the dose–response relationship was  (0.38    0.48  X  ) Y     0.032    3.6/[1    10 ]Gross effects were also seen at the organ and organismlevels. The EE 2  caused a significant reduction of body weightat the highest dose (Fig. 4A;  p    0.05 Dunnett’s a posterioritest after one-way ANOVA). Liver size, measured as hepa-tosomatic index, was significantly elevated in the four highestdoses (  p    0.05, Fig. 4B). At the two highest doses, hepa-tosomatic indices were almost twofold greater compared withcontrol lizards (  p    0.01).Plasma ALP was also determined in the preliminary dose–response and time course experiments using the same samplesanalyzed for plasma Vtg. In the single-injectiongroups,plasmaALP followed a similar trend as plasma Vtg, increasing withdose and time (Fig. 5A). In the multiple-injection treatmentgroups, plasma ALP was elevated at the time of first bloodcollection (Fig. 5B). However, unlike the plasma Vtg mea-surements in the same samples, ALP levels remained elevatedover the duration of the 27-d time course at all doses tested.In the dose–response experiment, plasma ALP followed asimilar trend as plasma Vtg, with an ED50 of 0.095 mg/kg  1414  Environ. Toxicol. Chem.  21, 2002 S.M. Brasfield et al.Fig. 6. Plasma alkaline-labile phosphate (ALP) concentrations in un-treated (sham injection and propylene glycol vehicle [PPG] controls)and ethinylestradiol-treated male western fence lizards. Plasma sam-ples were collected 15 d after a single intraperitoneal injection.Valuesare mean  standard error of the mean ([SEM][  n  7–8]). From thiscurve, an median effective dose (ED50) value of 0.095 mg/kg wascalculated for ALP induction.Fig. 7. Relative potencies of ethinylestradiol (EE 2 ) and 17  -estradiol(E 2 ) measured as plasma vitellogenin (Vtg) using enzyme-linked im-munosorbent assay (ELISA) and plasma alkaline-labile phosphate(ALP). Adult male western fence lizards from the San Joaquin Valley(CA, USA) were given a single intraperitoneal injection of 0.167 mg/ kg E 2  or EE 2  and blood was collected on day 15. Values are mean  standard error of the mean ([SEM] [ n  8]). Significant differencesare denoted by letters (a,  p  0.0011; b,  p  0.011) in Welch’s  t   test. (95% confidence limit: 0.018–0.68 mg/kg) (Fig. 6). The equa-tion describing the dose–response relationship was  (0.47    0.46  X  ) Y     0.13    1.0/[1    10 ]To further compare the two methods, plasma Vtg and ALPwere measured in six sample dilutions to generate a correlationrelating these two endpoints. The line revealed a strong linearcorrelation between these two techniques ( r   0.99). The equa-tion that describes this line was[Vtg]    0.23    [ALP]    0.0022Comparison of the potency of EE 2  with E 2  in this specieswas determined by administering both compounds at the EE 2 ED50 dose of 0.167 mg/kg. At this dose, EE 2  induced Vtg (  p   0.0011, Welch’s  t   test) and plasma ALP (  p    0.011) to asignificantly greater extent than E 2  (Fig. 7). DISCUSSION As an initial step in developing fence lizards as a laboratoryreptile model for endocrine-mediated toxicity, we character-ized dose–response and time course relationships for both plas-ma Vtg and ALP induction in adult male fence lizards exposedto EE 2 . Relatively little research has been conducted withfencelizards under laboratory conditions, and it is important to es-tablish baseline physiological responses in model species[5,8].Two responses were measured following treatments with EE 2 ,i.e., plasma Vtg using an antibody-capture ELISA and plasmaALP using a modified procedure of a commercially availablekit. The antibody-capture ELISA targets the protein usinghighly specific antibodies for  Sceloporus  Vtg and thus is adirect quantitative measure of Vtg. The plasma ALP methoddoes not measure Vtg directly but instead quantifies protein-associated phosphate groups. Previous research has shownthat, when present in the plasma, Vtg represents the majorityof the phosphoproteins [15].Few studies have examined the relative sensitivity and re-liability of ALP as a measure of Vtg expression in reptiles orother oviparous vertebrates. A previous study in fish [19] sug-gested that the sensitivity of ALP did not extend to low levelsof Vtg seen in nonreproductive females and juvenile animals.However, the detection limit for ALP in the present study was0.006   M phosphate (equivalent to 3.6 ng Vtg/ml plasma),and ALP was significantly elevated at the lowest dose admin-istered (0.0003 mg/kg). This indicates that ALP might be use-ful as a biochemical marker at low levels of estrogenic ex-posure in male oviparous vertebrates. To further compare thesetechniques, serial dilutions of plasma collected from an EE 2 -exposed male lizard were analyzed using both ELISA and ALP.A high concordance between plasma Vtg and ALP determi-nations was observed that extended to low levels of Vtg. Inaddition, our dose–response data further support the use of ALP as a substitute for Vtg ELISA, as both assays predictedsimilar ED50 values, 0.167 mg/kg for ELISA and 0.095 mg/ kg for plasma ALP. Thus, we believe the relationship describedbetween ALP and ELISA may be useful in future studies asa relatively rapid and technically less demanding alternativeto direct Vtg measurement with ELISA, at least at the levelsof Vtg measured in our study.Surprisingly, few studies have determined dose–responserelationships between treatment with EE 2  or other potent es-trogens and Vtg induction in oviparous vertebrates. To ourknowledge, there are no studies that have reported an ED50for Vtg induction in reptiles. In male Japanese quail ( Coturnixcoturnix japonica ), EE 2  did induce Vtg, measured as protein-bound phosphorus (PBP) [21], similar to the measure of ALPutilized in our study. Although PBP induction in quail did notappear to reach a maximum response, it appears that this avianspecies has a similar sensitivity to EE 2  as fence lizards. Infish, the relationship between EE 2  exposure and Vtg inductionhas been established; however, few dose–response studieshavebeen conducted. In a recent study in rainbow trout [22], ani-mals were given intravascular EE 2  injections and exhibited asimilar dose–response relationship for Vtg induction whencompared with fence lizards given i.p. injection. These studiessuggest that fence lizards exhibit a similar sensitivity to EE 2
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