The Psoralens Adversely Affect Reproductive Function in Male Wistar Rats

Reproductive Toxicology 15 (2001) 137–144 www.elsevier.com/locate/reprotox

The psoralens adversely affect reproductive function in male wistar rats

Moussa M. Diawaraa,*, Kathryn J. Chavezb, Doug Simplemana, David E. Williamsc, Michael R. Franklind, Patricia B. Hoyere

aDepartment of Biology, University of Southern Colorado, Pueblo, CO 81001, USA bDepartment of Comparative Environmental Toxicology, Cornell University, Ithaca, NY 14853, USA cDepartment of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331, USA dDepartment of Pharmacology and Toxicology, University of Utah, Salt Lake City, UT 84112, USA eDepartment of Physiology, University of Arizona, Tucson, AR 85721, USA

Received 3 October 2000; received in revised form 3 November 2000; accepted 23 December 2000

This research was supported, in part, by the National Institute of Environmental Health Sciences (NIH/NIEHS) grant ES00288 (to MMD), grant ADCRC #9808 (to PBH), and The University of Southern Colorado’s Faculty Scholarly Activity Grant.

Abstract The psoralens occur naturally in produce and are widely used in skin therapy. Studies show that 5-methoxypsoralen and 8-methoxypsoralen reduced birth rates in rats. We determined the effect of psoralens on reproductive function in male rats. Male Wistar rats were dosed daily with 5-methoxypsoralen or 8-methoxypsoralen (75 or 150 mg/kg, p.o.), or vehicle control. Treated males had significantly smaller pituitary glands, fewer sperm per ejaculate, and fewer sperm in the vasa defferentia and epididymides than controls. Dosing significantly elevated levels of testosterone and increased relative testis weight, but did not directly affect testicular weight. Females bred to dosed males required more time to become pregnant, and these males required more breeding attempts. The findings demonstrate the importance of determining the potential risk for infertility and/or birth defects in humans who are exposed to therapeutic, dietary, or occupational psoralens. © 2001 Elsevier Science Inc. All rights reserved.

Keywords: 5-Methoxypsoralen; 8-Methoxypsoralen; Birth rate; Testosterone; Sperm production; Birth defects; Reproductive behavior

1. Introduction thoxypsoralen (xanthotoxin or methoxsalen) in combination with UVA radiation, a procedure referred to as PUVA, The psoralens occur naturally as secondary metabolites remains the treatment of choice for skin disorders such as in various plant species including many fruits and vegeta- psoriasis and vitiligo [18–23]. The psoralens are also used bles [1–4]. The concentrations of psoralens found in pro- in the treatment of other skin conditions such as skin de- duce have occasionally been reported to be hazardous to pigmentation (leprosy and leucoderma), mycosis fungoides, human and animal health [3–5]. Plant-related health risks polymorphous dermatitis, and eczema. The United States associated with psoralens are due to handling and/or inges- National Toxicology Program tested 8-methoxypsoralen tion of psoralen-containing plant tissue [6–11]. These haz- (xanthotoxin or methoxsalen) for subchronic toxicity in ards are generally associated with photodermatitis, a skin male rats using oral administration [24]. The investigators reaction characterized by bulous eruption, pigmentation, found that the high doses of 8-methoxypsoralen (200 to 400 erythema, and vesicle formation [2,12]. mg/kg body weight) altered the histology of the liver, testes, The psoralens are known to be toxic to a wide range of and adrenals, reduced animal weight gain, increased relative organisms [3,4] and have been proven to be carcinogenic liver weight, and even caused death. The authors suggested and mutagenic [13–17]. Yet, oral administration of syn- that further investigations were needed to explain the male thetic forms of 5-methoxypsoralen (bergapten) and 8-me- genital organ atrophy observed during the study. A number of recent reports documented other potential risks associated with PUVA. Complications of burns re- * Corresponding author. Tel.: ϩ719-549-2813; fax: ϩ719-549-2732. ceived in a tanning salon by a patient under PUVA resulted E-mail address: [email protected] (M.M. Diawara). in death (2, and references therein). PUVA has been impli-

0890-6238/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S0890-6238(01)00118-6 138 M.M. Diawara et al. / Reproductive Toxicology 15 (2001) 137–144 cated in the increased incidence of malignant melanoma in doses/d: 0 mg/kg body wt for the vehicle control, 75 mg/kg patients, resulting in the death of some [19]. Studies by body wt for bergapten or xanthotoxin at 1250 ppm (range of Diawara et al. [25,26] showed that, in addition to the effects 64 to 116 mg/kg), and 150 mg/kg body wt for bergapten or reported by scientists at the National Toxicology Program xanthotoxin at 2500 ppm (range of 125 to 235 mg/kg). [24], oral administration of 8-methoxypsoralen and/or These concentrations were based on previous research [24– 5-methoxypsoralen to male and female rats also signifi- 27,29]. cantly reduced birth rate. Subsequent studies demonstrated As a clear indicator of the intrinsic toxic effect of the that the two compounds administered to females only (mat- psoralens [24,30], we observed during independent studies ed to undosed males) reduced levels of circulating blood that xanthotoxin exposure significantly reduced reproduc- estrogen, and the numbers of implantation sites, pups, and tive potential of male and female rats compared with control corpora lutea compared with the control group [27]. groups in the absence of UVA irradiation. Therefore, the In our previous studies, the reduction in the number of test animals were fed the experimental diet for eight weeks pups when only female rats were dosed with psoralens was on a 12:12 (L:D) cycle without UVA exposure during this less compared with the reduction when both sexes were experiment. dosed [26,27]. This finding suggests that the psoralens may have effects on both males and females. Thus, the present 1.3. Sperm output study was designed to determine whether exposure to the psoralens has a direct effect on reproductive function in Sperm counts were obtained by electroejaculation of the males. males [31] 37 and 57 d after the dietary treatments, i.e., when the test animals were 58 and 78 d old, respectively (the rats were 21 d old when the experiment was started). 2. Materials and Methods The male rat was secured on his back to a wooden board. A small probe (2 mm diameter, 1.5 cm long) lubricated with vaseline was inserted into the rectum to stimulate the pros- Experiment 1: Effect of psoralen dosing on sperm output in tate and seminal vesicles. The probe was operated on a male rats 110 v, 60-cycle alternating current, and the oscillator was run at 30 Hz with a maximum voltage of 3 v. Voltage was 1.1. Animals brought up slowly from 0 to 3 v and then brought down to 0 during a ten second-period. This treatment was repeated Male Wistar rats (21 days old) were obtained from until ejaculation occurred, which was observed for most test Charles River Laboratory (Charles River; WI/BR; Wilming- animals after approximately one minute of stimulation. As ton, MA), and were cared for at The University of Southern the animal ejaculated, the glans was gently squeezed to Colorado according to the guidelines of the Institutional remove all the semen. The ejaculate was collected in a Animal Care and Use Committee. microtube and enough eosin was added to bring the total volume to 1 mL. The sample was vortexed for 5 min and 1.2. Dietary treatments sperm were counted under light microscopy using a hemacytometer. On day 79, final weight was taken and animals Bergapten (5-methoxypsoralen) and xanthotoxin (8-me- were sacrificed with an overdose of CO . Relative liver thoxypsoralen) were obtained from Sigma Chemical Com- 2 weight was recorded. pany (Milwaukee, Wisconsin). Dietary treatments were prepared by incorporating the chemical into AIN-93G (ICN Experiment 2: Effect of the psoralens on reproductive Pharmaceuticals Inc., Costa Mesa, California) powdered organs diet as previously described [27]. The mixture was then The effect of psoralen dosing on the production of cir- blended into a warm agar/water solution for 5 min and culating testosterone, diameter of testicular tubules, and immediately dispensed into 30-mL plastic cups. The dietary weight of testes, seminal vesicles, prostate, epididymis, and treatments were allowed to solidify and kept in the refrig- pituitary glands was determined. erator until needed. The following treatments were used for bergapten and xanthotoxin: 0 (vehicle control), 1250, and 2500 ␮g chemical/g (ppm) diet. Each rat was housed in a 1.4. Animals and treatments separate cage (1 rat per cage; 10 rats per treatment group) and had access to the diet in non-spill stainless steel con- Animals and experimental conditions were the same as tainers and to deionized water. Based on the amount of for Experiment 1. However, test animals were kept on the agar/water solution added to the powdered diet mixture, the experimental diet for four weeks and three dietary groups average daily food intake, and daily weight of individual with ten males per group were used: 0 mg/kg body wt rats, the average daily dose of psoralens administered during (vehicle control), bergapten at 150 mg/kg body wt, and the study were determined to correspond to the following xanthotoxin at 150 mg/kg body wt. M.M. Diawara et al. / Reproductive Toxicology 15 (2001) 137–144 139

1.5. Tissue weights lating estrogen levels. By contrast with Experiment 1, in which sperm output was evaluated by electroejaculating

Animals were sacrificed by an overdose of CO2 30 d males, Experiment 3 evaluated sperm counts in the vas after the start of the experiment to observe psoralen-induced deferens and the epididymis at necropsy. subacute toxicity. The following observations were recorded: male body initial and final weight, weight of testes, 1.8. Animals and treatments seminal vesicles, prostate, epididymides, and pituitary glands. Circulating blood testosterone levels were also mea- Animals were purchased and maintained as described for sured in each animal at the time of tissue collection. Experiment 1. Two dietary groups with five males per group were used: 0 mg/kg body wt (vehicle control) and xantho- 1.6. Histologic examination toxin (Sigma Chemical Company, Milwaukee, WI) 150 mg/kg body wt. Females were obtained from Charles River At the time of tissue collection, testes were immediately Laboratory (Charles River; WI/BR; Wilmington MA) after fixed in 10% buffered formalin solution. Formalin-induced they had reached sexual maturity (45 d). Animals were shrinkage of tissue was assumed to be consistent for all maintained on regular chow diet and deionized water in treatment groups. For each testis, cross-sections (1 to 2 mm individual cages during the experiment. Females were ac- thick) were made and immersed in glycol methacrylate [32]. climated for a week before the start of breeding. Animals Sections (2.5 to 3 ␮m) obtained from these cross-sections were all appropriately labeled so that each of the five males were stained with periodic acid Schiff’s reagent and hema- in each dietary group was bred to three females; i.e., a total toxylin (PAS/H), and morphometrically examined by light of 15 females per group were tested. Vaginal lavage was microscopy. Tubule diameters of 100 nearly round seminif- checked daily (9:00 AM) in each female and used for erous tubules were measured for each testis using an ocular microscopic determination of estrous cycle stage [33]. On micrometer at 250ϫ. Diameter measurements were aver- the morning of proestrus, each female was transferred to the aged for each rat for statistical analysis. appropriate male cage at around 4:00 PM for overnight breeding. The feeder was removed from the male cage 1.7. Testosterone concentrations during the time the female was present. Females were removed from male cages early the next Testosterone concentrations were measured in serum and morning and the vaginal lavage was immediately examined testes according to the previously reported radioimmunoas- for the presence of sperm, which was used as evidence of say for 17␤-estradiol [27] with the following differences. mating and subsequent pregnancy. A female was bred again Testosterone antiserum (No. T4267) and testosterone were if sperm were not found. The date of breeding and number obtained from Sigma Chemical Company, St Louis MO. of breeding attempts (i.e., the number of days on which 3H-Testosterone was purchased from Amersham (80 Ci/ males were caged with females) were recorded. Dams and mmol). Testicular homogenates and sera were extracted fetuses were sacrificed two days before expected parturition with diethyl ether, and recovery was monitored in each (Day 19 of pregnancy). All males were sacrificed 79 d after sample. The sensitivity was 23.9 pg/mL, the intraassay the start of experiment after pregnancy had been established coefficient of variation was 3.54%, and interassay coeffi- for each of the females in the study. cient of variation was 8.10%. To simulate conditions of the experiment in which both males and females had been dosed [26] and the experiment Experiment 3: Effect of male dosing on reproductive func- in which only females were dosed [27], all males (control tion and treated) received UVA irradiation using fluorescent This experiment used dosed males and undosed females lamps (40-W Philips F Black Light, burn in 20,000 h, Light to investigate effects on mating behavior and success, in- Bulb Supply, Denver, Colorado) adjusted at approximately cluding the number of breeding attempts; numbers of im- 40 cm above the rat cages to produce 300 ␮W/cm2 long- plantation sites, fetuses, empty spaces, and reabsorbed fe- wave UVA radiation. The exposure to UVA lasted for 45 tuses; and placental, ovarian, and uterine (full and empty) min/d during the daytime portion of the 12:12 (L:D) light weight. The effect on individual fetuses resulting from mat- cycle for the duration of the experiment. Females were on ing undosed females with dosed males was also determined. the same 12:12 (L:D) cycle but were not exposed to UVA. Another goal of Experiment 3 was to compare the level of circulating and testicular testosterone to determine the po- 1.9. Northern blot analysis tential impact of the psoralens on steroidogenesis. The psoralens induced mRNAs of cytochrome (CYP) 1A1 in female RNA was prepared from frozen male liver samples as rats during our previous studies [27]. Another objective of previously described [27]. Briefly, RNA from each sample Experiment 3 was a further exploration of xanthotoxin- was subjected to electrophoresis. Blots were washed at high induced modulation of the CYP1A family, an observation stringency and exposed to autoradiographic film. Autorad that is consistent with psoralen-induced reduction in circu- band intensity was determined by scanning densitometry 140 M.M. Diawara et al. / Reproductive Toxicology 15 (2001) 137–144

(HP ScanJet II 2cx/T Flat Bed Scanner, NIH Image Version 1.54 - Available from the Internet by Anonymous FTP from zippy.nimh.nih.gov). The cDNA probes utilized were ϩ1511 to ϩ2202 for rat CYP1A1 [34], ϩ28 to ϩ810 for rat UGT1A6 [35], and Ϫ18 to ϩ728 for rat UGT2B1 [36]. Plasmids containing cDNA probes were kindly provided to MRF by Dr. J. Ritter, Medical College of Virginia, Virginia Commonwealth University, Richmond.

1.10. Evaluation of females

The body weight of each female was determined upon arrival, three weeks later (before any pregnancy-related weight gain), and on the day of sacrifice. At the time of sacrifice by CO2 overdose, the weights of full and empty uteri, and individual fetuses were recorded. The number of reabsorbed fetuses, implantation sites, corpora lutea, devel- oped fetuses, and empty spaces, and the size (length and Fig. 1. Effect of dietary psoralens bergapten (B) and xanthotoxin (X) on girth) of individual fetuses, fetal deformities, and discolor- sperm output in Wistar rats. *Statistically different from the control group ation were also recorded. The number of breeding attempts at the 5% level for each variable according to Duncan’s multiple range test. Bars are standard errors. (Control n ϭ 5, xanthotoxin n ϭ 5). (number of days on which males were caged with females) before successful pregnancy for each female, the frequency of presence and absence of sperm in the vaginal lavage after say coefficient of variation was 6.6%, with a sensitivity of an overnight breeding attempt, the number of pseudopreg- 180 pg/tube. nancies, and the number of days to pregnancy were deter- Statistical methods mined for each female. For each of the three experiments, data were analyzed using General Linear Model of ANOVA [28]. For each 1.11. Evaluation of males variable, means statistically different were separated at the 5% level using Fisher’s Protected LSD test. For males, the initial and final animal weight and weights of pituitary glands, testes, epididymides, vasa deferentia, and livers were recorded. Previous researchers reported that 3. Results sperm production in Fisher 344 rats could not be reliably determined based on epididymal sperm count alone [37]. Thus, sperm were collected from the vas deferens and cauda Experiment 1: Effect of psoralen dosing on sperm produc- epididymis separately, and caput plus corpus epididymis tion in male rats combined. For the vas deferens, sperm were collected by Bergapten and xanthotoxin (together or alone) reduced flushing the organ with 1 mL PBS in a small centrifuge tube, sperm counts/ejaculate in a dose-dependent manner (Fig. 1). which was vortexed for 1 min. A 1:10 dilution was then When counts were taken 37 d after the start of the experi- made in eosin solution and sperm counts were obtained ment (i.e., when rats were 58 d old), all psoralen treatments using a hemacytometer. For the epididymis (cauda or corpus except for the low dose of xanthotoxin (75 mg/kg body wt) plus caput combined), organs were sliced in 1 mL PBS significantly reduced sperm output compared with controls. using scissors and the solution was vortexed for 1 min at 6 Males administered the high dose of xanthotoxin failed to g. As with the vas deferens, a hemacytometer was used to produce any detectable sperm despite repeated efforts using take counts from a 1:10 sperm/PBS:eosin dilution. Previous electroejaculation at 58 d of age. When the rats were elec- workers have collected sperm from the vas deferens or the troejaculated at 78 d of age, all test animals ejaculated epididymis [38,39]. sperm, but counts were significantly lower in all treated Circulating follicle stimulating hormone (FSH) levels animals except the low dose of bergapten. Body weight gain were measured in serum by radioimmunoassay using a kit was reduced in all animals receiving xanthotoxin or the high (RPA 550; Amersham, Piscataway NJ). All samples were dose of bergapten compared with the control group (Table assayed at once. The intrassay coefficient of variation was 1). However, only the high dose of bergapten and the 7.9%, with a sensitivity of 185 pg/tube. Circulating lutein- combined diet caused significant reduction in body weight izing hormone (LH) levels were measured in serum by change as a percentage of control body weight change. radioimmunoassay using a kit (RPA 552. Amersham, Pis- Relative liver weight was significantly increased by the high cataway NJ). All samples were assayed at once. The intraas- doses as well as the combination of the two compounds. M.M. Diawara et al. / Reproductive Toxicology 15 (2001) 137–144 141

Table 1 Effects of dietary 5-methoxypsoralen (bergapten) and 8-methoxypsoralen (xanthotoxin) on liver somatic index and weight gain in male Wistar rats

Treatment Relative liver weight* Body weight change** Body weight change (mean Ϯ sem) (g, mean Ϯ sem) (% of control weight change) Control 4.67 Ϯ 0.44a 290.5 Ϯ 5.5bc Reference Bergapten 75 mg/kg body wt 5.63 Ϯ 0.14abc 307.5 Ϯ 12.5c 5.9 Bergapten 150 mg/kg body wt 6.80 Ϯ 0.08cd 259.4 Ϯ 8.3a Ϫ10.7d Xanthotoxin 75 mg/kg body wt 5.41 Ϯ 0.33ab 280.5 Ϯ 15.0abc Ϫ3.4 Xanthotoxin 150 mg/kg body wt 6.20 Ϯ 0.54bcd 271.8 Ϯ 18.5ab Ϫ6.4 Bergapten ϩ xanthotoxin 7.07 Ϯ 0.33d 249.1 Ϯ 3.8a Ϫ14.2d (75 mg/kg body wt each)

n ϭ 10/treatment group. * (Liver wt/body wt) ϫ 100. ** Body weight change ϭ ﬁnal wt ? initial wt. abc Means within a column followed by the same letter are not statistically different at the 5% level according to Fisher’s Protected Least Signiﬁcant Difference test. d Signicantly different from control, P Ͻ 0.05.

Experiment 2: Effect of the psoralens on reproductive sites (14.3 control vs. 14.7 xanthotoxin), fetuses (13.4 con- organs trol vs. 13.6 xanthotoxin), and reabsorbed fetuses (0.09 In this experiment, oral administration of bergapten and control vs. 0.46 xanthotoxin) were not different. Full uterine xanthotoxin significantly reduced weights of seminal vesi- weight (56.5 g control vs. 55.3 g xanthotoxin), empty uter- cles, prostates, epididymides, and pituitary glands compared ine weight (4.7 g control vs. 4.9 g xanthotoxin), and ovarian with untreated animals (Table 2). Although the psoralens weight (0.22 g control vs. 0.20 g xanthotoxin) were also not did not significantly affect total testicular weight, relative affected. testicular weights were increased significantly by xantho- The numbers of sperm collected from the vasa deferentia toxin treatment. Testicular tubule diameter showed no sig- and the epididymides in treated males were significantly nificant psoralen-induced adverse effect (Table 2). reduced compared with controls (Table 4). Dosing reduced the size of the pituitary gland and increased relative testis Experiment 3: Effect of male dosing on reproductive func- weight, but did not affect the weights of seminal vesicle tion (0.72 g control vs. 0.66 g xanthotoxin), prostate (0.28 g As compared with control, treated males required a sig- control vs. 0.22 g xanthotoxin), whole epididymis (1.12 g nificantly greater number of breeding attempts to establish control vs. 1.11 g xanthotoxin), or vas deferens (0.22 g pregnancy, associated with a greater frequency of an ab- control vs. 0.20 g xanthotoxin). The levels of serum and sence of sperm in the vaginal smear following an overnight testicular testosterone were significantly higher in treated breeding attempt (Table 3). males (Table 4). There was no effect of treatment on epi- The girth of litters from females bred to xanthotoxin- didymal testosterone, serum LH, or serum FSH (Table 4). treated males was significantly smaller compared with con- All dosed animals had a significant induction of mRNA’s trols, but there were no differences in the other fetal param- for CYP1A1, UGT1A6, and UGT2B1 in livers compared eters as a result of treatment (Table 3). The number of with the control animals (Fig. 2). placentas (13.6 control vs. 14.4 xanthotoxin), implantation Psoralen-induced teratogenic effects were observed in

Table 2 Effect of the dietary 5-methoxypsoralen (bergapten, 150 mg/kg) and 8-methoxypsoralen (xanthotoxin, 150 mg/kg) on reproductive organs in male Wistar rats

Treatment Pituitary wt Seminal Prostate wt Testis wt (g) Relative Epidydimal wt Testicular Serum (mg) vesicle wt (g) testis wt* (g) tubule testosterone (g) diameter (ng/mL) (␮m) Control 9.0 Ϯ 1.0* 0.70 Ϯ 0.06a 0.23 Ϯ 0.03a 1.3 Ϯ 0.05a 0.5 Ϯ 0.01a 0.39 Ϯ 0.02a 272.3 Ϯ 3.2a 1.268 Ϯ 0.11a Bergapten 8.0 Ϯ 0.9b 0.37 Ϯ 0.07b 0.13 Ϯ 0.02b 1.3 Ϯ 0.07a 0.6 Ϯ 0.03a 0.34 Ϯ 0.02b 264.3 Ϯ 5.6a 1.600 Ϯ 0.09a Xanthotoxin 6.0 Ϯ 1.0b 0.27 Ϯ 0.04b 0.10 Ϯ 0.03b 1.2 Ϯ 0.06a 0.7 Ϯ 0.04b 0.23 Ϯ 0.01b 259.5 Ϯ 6.1a 1.237 Ϯ 0.14a

Data expressed as mean Ϯ sem, n ϭ 10/treatment group. ab Means within a column followed by the same letter are not statistically signiﬁcant at the 5% level according to Fisher’s Protected Least Signiﬁcance test. * (Testis wt/Body wt) ϫ 100. 142 M.M. Diawara et al. / Reproductive Toxicology 15 (2001) 137–144

Table 3 Effect of dosing male Wistar rats with 8-methoxypsoralen (xanthotoxin) (150 mg/kg) on reproductive efﬁciency in females

Variable Control Xanthotoxin P value (n ϭ 14) (n ϭ 15) Number of breeding attempts 2.2 Ϯ 0.3 7.1 Ϯ 1.0 0.0004 Number of times no sperm were found in the vaginal smear 1.2 Ϯ 0.3 5.2 Ϯ 0.9 0.0011 after overnight breeding attempt Number of days to pregnancy (from day of ﬁrst breeding 20.0 Ϯ 2.9 48.0 Ϯ 4.9 0.0001 attempt) Placenta weight/litter (g) 0.53 Ϯ 0.02 0.50 Ϯ 0.03 0.3877 Fetal parameters/litter weight (g) 2.31 Ϯ 0.05 2.07 Ϯ 0.15 0.1498 length (mm) 30.18 Ϯ 0.58 29.46 Ϯ 1.21 0.5681 girth (mm) 11.38 Ϯ 0.14 10.69 Ϯ 0.33 0.0508 malformed fetuses (n) 0.00 Ϯ 0.00 0.15 Ϯ 0.10 0.1795 discolored fetuses (n) 0.00 Ϯ 0.00 0.85 Ϯ 0.44 0.0893

Data expressed as mean Ϯ sem. two of 177 fetuses from treated males. These fetuses were (oral administration of psoralen drugs in the presence of totally deformed or had hind limbs missing. In addition, 11 UVA to treat psoriasis) prior to pregnancy. The authors out of the 177 fetuses from dosed males had marked dis- suggested that this effect was not due to maternal smoking. colored patches on the abdomen or were pale (almost Because it was unclear whether the disease itself (psoriasis) white), suggesting a potential cardiovascular effect. No de- was implicated in the observed low birth weight, the authors formities or discolorations were observed in 174 fetuses concluded that PUVA did not have significant mutagenic or from controls. The litter incidence of external malforma- teratogenic effects. Because females were not subjected to tions or discoloration in the psoralens group was not sig- PUVA in the present study, our findings of comparable fetal nificantly different from the control (Table 3). weight/litter from PUVA-exposed and control sires does not specifically address the Gunnarskog observation. However, the reduced fetal girth observed during the current study 4. Discussion suggests that the psoralens might have been implicated in results observed by Gunnarskog and colleagues [40]. Gunnarskog et al. [40] reported a “marked increase in Dosing significantly reduced weights of pituitary glands, low-birth weight infants” born to women exposed to PUVA prostate, seminal vesicles, and epididymides, and increased relative testis weight during Experiment 2 (Table 2). It is Table 4 unclear why some accessory glands differed significantly in Effect of dosing with 8-methoxypsoralen (xanthotoxin) (150 n weight during Experiment 2, but not during Experiment 3. reproductive function in male Wistar rats The main differences between the two studies are the dura- Variable Control Xanthotoxin P (n ϭ 3-5) (n ϭ 3-5) value

Sperm numbers (ϫ106) Per vas deferens 88.52 Ϯ 5.16 63.81 Ϯ 2.24 0.0299 Per epididymis 111.72 Ϯ 8.36 76.09 Ϯ 3.34 0.0167 Per g epididymis 198.21 Ϯ 13.76 153.76 Ϯ 6.170 0.0421 Weight Pituitary gland (mg) 14.0 Ϯ 1.0 10.0 Ϯ 1.0 0.0108 Testis (g) 1.88 Ϯ 0.03 2.00 Ϯ 0.7 0.1715 Relative testis weight* 0.364 Ϯ 0.14 0.512 Ϯ 0.14 0.0001 Testosterone (ng/mL) Serum 1.22 Ϯ 0.11 1.76 Ϯ 0.13 0.0143 Testis 11.79 Ϯ 2.38 22.33 Ϯ 2.56 0.0168 Epididymis 2.78 Ϯ 1.21 4.08 Ϯ 0.77 0.3915 Ratio serum testosterone/ 0.115 Ϯ 0.017 0.086 Ϯ 0.017 0.2655 testis testosterone Serum gonadotropins (ng/mL) Luteinizing hormone 2.45 Ϯ 0.48 3.78 Ϯ 0.37 0.0696 Follicle stimulating 2.01 Ϯ 0.57 3.40 Ϯ 0.48 0.0988 Fig. 2. Effect of dietary psoralens bergapten (B) and xanthotoxin (X) on hormone liver mRNAs of CYP1A1, UGT1A6, and UGT2B1 in male Wistar rats. *Statistically different from the control group at the 5% level for each Data are mean Ϯ sem. variable according to Duncan’s multiple range test. Bars denote standard * (Testis wt/body wt) ϫ 100. errors. (Control n ϭ 5, xanthotoxin n ϭ 5). M.M. Diawara et al. / Reproductive Toxicology 15 (2001) 137–144 143 tion of the dosing period (8 weeks for experiment 2 versus result from a direct effect on the male, specifically the 11 weeks for experiment 3) and the exposure to UVA sperm. Since copulatory behavior was not visually moni- radiation during Experiment 3. The increase in relative tored, it is also possible that treatment could have depressed testicular weight supports the possibility that an increase in libido, resulting in increased numbers of breeding attempts. circulating LH caused a trophic effect and increased testic- The mechanism of xanthotoxin-induced male reproductive ular tissue mass, although such an increase was shown in the toxicity remains unclear and requires further investigation; current study only at a P value Ͻ0.07. Additionally, the however, a direct toxic effect on fertilization, implantation, increased circulating and testicular testosterone (Table 4) and/or placental development cannot be ruled out. These would also result from increased LH effects on steroidogen- findings demonstrate the importance of determining the esis. No differences were found in the weight of the vasa potential risk for infertility and/or birth defects in humans deferentia of treated and untreated animals during Experi- who are exposed to therapeutic (PUVA), dietary (produce ment 3; however, sperm counts from these organs were consumption), or occupational (agricultural or industrial) significantly reduced in dosed males (Table 4). Because psoralens. testis weight was not significantly affected, it appears that the brain and the hypothalamic-pituitary axis may be more susceptible to the effect of the psoralens than is the testis. Acknowledgment This possibility was recently suggested by Turner et al. [41] after they found similar results while studying the effect of The authors thank Stewart Garner, Stacy McCrorry, a nonsteroidal aromatase inhibitor, anastrozole, on pituitary Brenda Boese and Samuel L. Marion for their assistance in and testicular function and fertility in male rats. the laboratory, Dr. Paul Kulkosky for his support, Dr. Davi Our previous studies demonstrated that xanthotoxin Gonzales for his technical advice. We are also grateful to compromises the development of ovarian follicles in rats Dr. Kim Boekleheide and Dr. Heidi Schoenfeld of the [42], which is consistent with its observed reduction in Department of Pathology and Laboratory Medicine at circulating 17␤-estradiol levels [27]. Sertoli cells are the Brown University for their assistance with the histological male equivalent of female granulosa cells; their main func- examinations. This research was supported, in part, by the tion is to protect and nourish the growing sperm. Although National Institute of Environmental Health Sciences (NIH/ this similarity of function does not make these cells equiv- NIEHS) grant ES00288 (to MMD), grant ADCRC #9808 alent from a toxicologic standpoint, the potential impact of (to PBH), and The University of Southern Colorado’s Fac- xanthotoxin on Sertoli cells should be investigated. ulty Scholarly Activity Grant. Induction of CYP1A1 is of interest because such an induction would lower circulating levels of estrogen in women, and inducers of this isoenzyme have been proposed References as chemopreventive agents for estrogen-dependent cancer [43]. Both CYP1A1 and CYP1A2 are effective in 2-hy- [1] Murray RDH, Mendez J, Brown SA, editors. The Natural Coumarins. droxylation of estrogen [44]. Gwang [30] also found that ip Chichester: J. Wiley and Sons, 1982. administration of xanthotoxin to male Sprague-Dawley rats [2] Beier RC. Natural pesticides and bioactive components in foods. Rev induced liver CYP1A, CYP2B, and UGT2B1 mRNAs, pro- Environ Contam Toxicol 1990;113:47–129. teins, and catalytic activities in a dose-dependent manner [3] Berenbaum MR. Coumarins. In: Rosenthal GA, Berenbaum MR, editors. Herbivores Their Interactions with secondary plant metabo- [35]. The induction of UGT1A6 mRNA with CYP1A1 in lites, the chemical participants, Vol. I. Academic Press, New York: the current study, as well as our previous studies [27], is an 1991;6:221–49. indication that xanthotoxin may be a direct or indirect li- [4] Diawara MM, Trumble JT. Linear Furanocoumarins. In: D’Mello gand for the Ah-receptor and modulate the AhR/Arnt signal JPF, editor. Handbook on plant and fungal toxicants. CRC Press, Inc., transduction pathway. The induction of UGT2B1, however, Boca Raton: 1997;12:175–89. [5] Ivie GW, Holt DL, Ivey MC. Natural toxicants in human foods: suggests that xanthotoxin can also act through other signal- psoralens in raw and cooked parsnip root. Science 1981;213:909–10. ing pathways. [6] Austad J, Kavli G. Phototoxic dermatitis caused by celery infected by The observations reported here provide evidence for pso- Sclerotinia sclerotiorum. Contact Dermatitis 1983;9:448–51. ralen-induced reproductive effects in male rats. These ef- [7] Berkley SF, Hightower AW, Beier RC, Fleming DW, Brokopp CD, fects were not evaluated by the National Toxicology Pro- Ivie GW, Broome CV. Dermatitis in grocery workers associated with high natural concentrations of furanocoumarins in celery. Ann Intern gram research team [24] and differ from those reported in Med 1986;105:351–5. our study of female-only dosed animals [27]. During the [8] Seligman PJ, Mathias CG, O’Malley MA, Beier RC, Fehrs LJ, Serril current study, females were not dosed during the breeding WS, Halperin WE. Photodermatitis from celery among grocery store experiment (Experiment 3) and were only potentially ex- workers. Arch Dermatol 1987;123:1478–82. posed to the psoralen through seminal fluid. Consequently, [9] Fleming D. Dermatitis among grocery workers associated with high natural concentrations of furanocoumarins in celery. Allergy Proc it is important to note that the high number of breeding 1990;11:125–7. attempts, the reduced fetal girth, and possibly the abnormal- [10] Ljunggren B. Severe phototoxic burn following celery ingestion. ities observed in offspring of females mated to treated males Arch Dermat 1990;126:1334–6. 144 M.M. Diawara et al. / Reproductive Toxicology 15 (2001) 137–144

[11] Finkelstein E, Afek U, Gross E, Aharoni N, Rosenberg L, Havely S. tion and identification of metabolites. J Pharmacol Exp Ther 1986; An outbreak of phytophotodermatitis due to celery. Internat J Dermat 236:364–73. 1994;33:116–8. [30] Gwang JH. Induction of rat hepatic cytochrome P4501A and P4502B [12] Musajo L Rodighiero G. The skin-photosensitizing furanocoumarins. by the methoxsalen. Cancer Lett 1996;108:115–20. Experientia 1962;18:153–200. [31] Lawson RL, Krise GM, Sorensen AM. Electroejaculation and eval- [13] Ashwood-Smith MJ, Poulon GA, Barker M, Mildenberger M. 5-Me- uation of semen from the albino rat. J Appli Physiol 1967;22:174–6. thoxypsoralen, an ingredient in several suntan preparations, has le- [32] Chapin RE, Ross MD, Lamb JC. Immersion fixation methods for thal, mutagenic and cleistogenic properties. Nature 1980;285:407–9. glycol methacrylate-embedded testes. Toxicol Pathol 1984;12:221–7. [14] Roelandts R. Mutagenicity and carcinogenicity of methoxypsoralen [33] Bennett JP, Vickery BH. Rat and mice. In: Hafez ESE, editor. Re- plus UV-A. Arch Dermatol 1984;120:662–9. production and breeding techniques for laboratory animals. Philadel- [15] Young AR. Photocarcinogenicity of psoralens used in PUVA treat- phia 1970;17:299–15. ment-present status in mouse and man. J Photochem & Photobiol [34] Yabusaki Y, Shimizu M, Murakami H, Nakamura K, Oeda K, Oh- B-Biol 1990;6:237–47. kawa H. Nuceotide sequence of a full-length cDNA encoding for [16] Bauluz C, Paramio JM, de-Vidania R. Further studies on the lethal 3-methylcholnathrene-induced rat liver cytochrome P-450MC. Nu- and mutagenic effects of 8-methoxypsoralen-induced lesions on plas- cleic Acid Res 1984;12:2929–38. mid DNA. Cell Mol Biol 1991;37:481–3. [35] Iyanagi T, Haniu M, Sogawa K, Fujii-Kuriyama Y, Watanabe S, [17] Yang SC, Lin JG, Chion CC, Chen LY, Yang JL. Mutation specificity Shively JE, Anan KF. Cloning and characterization of cDNA encod- of 8-methoxypsoralen plus two doses of UVA radiation in the hprt ing for 3-methylcholanthrene-inducible rat mRNA for UDP-glucuro- gene in diploid human fibroblasts. Carcinogenesis 1994;15:201–6. nyltransferase. J Bio Chem 1986;261:6119–125. [18] Stern RS, Thibodeau LA, Kleinerman RA, Parrish JA, Fitzpatrick TB. [36] Mackenzie PI. Rat liver UDP-glucuronosyl transferase. Sequence and Risk of cutaneous carcinoma in patients treated with oral methoxsalen expression of a cDNA encoding a phenobarbital-inducible form. photochemotherapy for psoriasis. N Engl J Med 1979;300:809–13. J Biol Chem 1986;261:6119–6125. [19] Stern RS, Nichols KT, Vakeva LH. Malignant melanoma in patients [37] Blazak WF, Ernst TL, Stewart BE. Potential indicators of reproduc- treated for psoriasis with methoxsalen (psoralen) and ultraviolet ra- tive toxicity: testicular sperm production and epididymal sperm num- diation (PUVA). N Engl J Med 1997;336:1041–5. ber, transit time, and motility in Fisher 344 rats. Fundamental Appl [20] Brickl R, Schmid J, Koss FW. Clinical pharmacology of oral psoralen Tox 1985;5:1097–1103. drugs. Photodermatology 1984;1:174–8. [38] Klinefelter GR, Gray LE, Suarez JD. The method of sperm collection [21] de Wolff FA, Thomas TV. Clinical pharmacokinetics of methoxsalen significantly influences sperm motion parameters following ethane and other psoralens. Clin Pharmacokinetics 1986;11:62–5. dimethanesulphonate administration in the rat. Reprod Toxicol 1991; [22] Pathak MA, Fitzpatrick TB. The evolution of photochemotherapy 5:39–44. with psoralens and UVA (PUVA): 2000 BC to 1992 AD. Photochem Photobiol B 1992;14:3–33. [39] Slott VL, Suarez JD, Perreault SD. Rat sperm motility analysis: [23] Sternberg S. Psoriasis balm boosts melanoma risk. Science News methodological considerations. Reprod Toxicol 1991;5:449–58. 1997;151:272. [40] Gunnarskog JG, Bengt Kallen AJ, Lindelof BG, Sigurgeirsson B. [24] Dunnick JK, Davis WE, Jorgenson TA, Rosen VJ, McConnell EE. Psoralen photochemotherapy (PUVA) and pregnancy. Arch Dermatol Subchronic toxicity in rats administered oral 8-methoxypsoralen. 1993;129:320–3. National Cancer Institute Monographs 1984;66:91–5. [41] Turner KJ, Morley M, Atanossova N, Swnason ID, Sharp RM. Effect [25] Diawara MM, Allison T, Kulkosky P, Williams DE. Psoralen-in- of chronic administration of an aromatase inhibitor to adult male rats duced growth inhibition in Wistar rats. Cancer Letters 1997a;114: on pituitary and testicular function and fertility. J Endocrinol 2000; 159–60. 164:225–38. [26] Diawara MM, Allison TG, Kulkosky P, McCrory S, Martinez LA, [42] Diawara MM, Hoyer PH, Chavez KJ, Williams DE. 8-Methoxypso- Williams DE. Mammalian toxicity of bergapten and xanthotoxin, two ralen (xanthotoxin or methoxsalen) compromises ovarian follicle de- compounds used in skin photochemotherapy. J Natural Toxins 1997b; velopment in rats. In: Johnson IT, Fenwick GR, editors. Dietary 6:183–92. anticarcinogens and mutagens: chemical and biological aspects. Cam- [27] Diawara MM, Chavez KJ, Hoyer PB, Williams DE, Dorsch J, Kul- bridge: Royal Society of Chemistry, 2000. p. 361–5. kosky P, Franklin MR. A new group of ovarian toxicants: the psor- [43] Bradlow HL, Sepcovic DW, Telang NT, Osborne MP. Indole-3- alens. J Biochem Mol Toxicol 1999;13:195–203. carbinol. A novel approach to breast cancer prevention. Ann N Y [28] SuperANOVA. SuperANOVA, Berkeley: Abacus Concepts Inc., Acad Sci 1995;768:180–200. 1989. [44] Jellinck PH, Forkert PG, Riddick DS, Okey AB, Michnovicz JJ, [29] Mays DC, Rogers SL, Guiler RC, Sharp DE, Hecht SG, Staubus AE, Bradlow HL. Ah receptor binding properties of indole carbinols and Gerber N. Disposition of 8-methoxypsoralen in the rat: methodology induction of hepatic estradiol hydroxylation. Biochem Pharmacol for measurement, dose-dependent pharmacokinetics, tissue distribu- 1993;45:1129–36.