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Reproduction of the tundra vole (Microtus oeconomus) with dietary phytosterol supplement
Food and Chemical Toxicology 42 (2004) 945–951 www.elsevier.com/locate/foodchemtox
Reproduction of the tundra vole (Microtus oeconomus) with dietary phytosterol supplement P. Nieminen*, A.-M. Mustonen, P. Pa¨iva¨la¨inen, J.V.K. Kukkonen University of Joensuu, Department of Biology, PO Box 111, FIN-80101 Joensuu, Finland Received 6 August 2003; accepted 7 February 2004
Abstract Phytosterols (PS) are plant-derived compounds with estrogenic activity in vitro and beneficial effects on the serum lipid profile in vivo. In nature, PS exposure can derive from pulp mill effluents. The effects of a pulp-mill derived PS mixture on the reproduction, endocrine variables and enzyme activities of the tundra vole (Microtus oeconomus) were investigated in a two-generation study. The cumulative food intake of PS-treated females was higher than in the control group supporting previous results on the effects of PS on food consumption in rodents. 85% of the PS treated pairs reproduced, but the figure was only 60% for the control pairs. The plasma and testicular testosterone concentrations were lower in the adult PS males, but the PS-treated male offspring had higher testicular testosterone concentrations than their controls. In the female offspring, the liver lipase activity was higher in the PS-treated group, which could be a result of decreased cholesterol absorption in the gut. Chronic PS treatment increased the reproduction probability of the species and had a potential effect on the sex steroid hormones of maturing offspring, which could have applications in environmental monitoring. # 2004 Elsevier Ltd. All rights reserved. Keywords: Microtus oeconomus; Reproduction; Sitosterol; Testosterone; Tundra vole
1. Introduction The beneficial effects of phytosterols (PS) and phytostanols to lower elevated total and low density lipoprotein cholesterol levels have been thoroughly documented (Drexel et al., 1981; Miettinen et al., 1995; Jones et al., 2000). Due to this, PS are added to margarines at 30 mg kg1 day1 (Hallikainen et al., 2000). Pulp mill effluents are a source of PS in nature. PS cause reduced sex steroid levels and gonad size in fish (Van der Kraak et al., 1992; Mellanen et al., 1996). b-Sitosterol induces vitellogenin gene expression in the male rainbow trout (Oncorhynchus mykiss) and it also has estrogenic activity in T-47D breast cancer cells (Mellanen et al., 1996). In rats, subcutaneous b-sitosterol at doses of 0.5–5 mg kg1 day1 reduces sperm count and testicular weight (Moghadasian, 2000). However, PS mixtures do not cause estrogenicity at 0–500 mg kg1 day1 measured with effects on uterine weight of immature female rats * Corresponding author. Tel.: +358-13251-3576; fax: +358-132513590. E-mail address: [email protected].fi (P. Nieminen). 0278-6915/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2004.02.006
(Baker et al., 1999). In European polecats (Mustela putorius), PS exposure leads to increases in the circulating estradiol and thyroid hormone levels (Nieminen et al., 2002a). This may be caused by PS being used as precursors of sex steroids in gonads (Moghadasian, 2000). The tundra vole (Microtus oeconomus, Pallas, 1776) is a small rodent with a circumpolar distribution (Tast, 1966). Its diet consists of diverse leaves, flowers, seeds and stalks of different grass or sedge species. The species is a seasonal long-day breeder and reproduction can be induced in laboratories by exposing the animals to long daylength. Previously it has been documented in dose– response studies on microtines that a two-week exposure to PS at 5 mg kg1 day1 increases the circulating testosterone concentrations of male voles and the liver glycogen phosphorylase activities of both sexes (Nieminen et al., 2003). As a regionally abundant circumpolar species the tundra vole also has potential as a bioindicator of possible endocrine disruption in arctic and boreal nature. The aim of the present study was to investigate the effects of PS on the reproduction, endocrinology and parameters of intermediary metabolism of adult voles and on the physiology of their F1 offspring.
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2. Materials and methods 2.1. Animals and exposure Forty-six tundra voles (Microtus oeconomus; 23 males, 23 females, age 91 6 days; sexual maturity reached at the age of 70–80 days) from the breeding colony of the University of Joensuu were selected for the study. The animals were housed singly in solid bottom plastic cages (Makrolon: 422215 cm) with wood shavings for bedding and free access to water and a pelleted diet (Avelsfoder fo¨r ra˚tta och mus R36: protein 18.5%, fat 4.0%, energy content 1260 kJ 100 g1, Lactamin, Stockholm, Sweden). The animals were assigned randomly into two experimental groups. The control group (10 pairs) receiving the regular diet and the PS group (13 pairs). Originally, the number of pairs in both experimental groups was the same (14 control and 14 PS treated pairs). Due to difficulties in the determination of sex of juvenile voles some of the formed pairs were female– female pairs and these were excluded from the analyses. The PS group received peroral PS (Ultra-sitosterol1: 88.7% b-sitosterol+b-sitostanol, 9.0% campesterol +campestanol, 0.9% artenols; mixture derived from pulp mill effluents by UPM Kymmene, Kaukas, Lappeenranta, Finland) mixed into the normal food of the animals at 5 mg kg1 day1. This is a single dose study; other doses were not needed as the dose is based on previous dose-response studies showing significant effects on microtine voles at this fairly low dose of PS, such as increased plasma testosterone and estradiol concentrations in male voles with an opposite trend of plasma luteinizing hormone concentrations (Nieminen et al., 2003). At a higher dose (50 mg kg1 day1) there was a return to the level of control animals. Yet 5 mg kg1 day1 is a relevant dose, as it is the recommended dose of PS for several natural remedies and can be also encountered in nature. After four weeks, the animals were paired, allowed to mate and reproduce freely. After parturition, the male was removed from the cage. Food intake was measured weekly by weighing the amount of food left uneaten by an individual vole or a pair at the end of each study week. Food intake during mating period represents the average of the male–female pair and during the lactating period mostly that of the lactating female, but closer to the weaning age of three weeks, the young voles also consumed some of the pellets. The body mass (BM) of all the animals was measured weekly, and the BMs of the offspring at the age of 1, 7 and 14 days. The Animals Care and Use Committee of the University of Joensuu approved all procedures. Both the parents and the offspring were sacrificed three weeks after parturition (weaning age of the offspring). The animals were euthanized quickly with diethyl ether and their BM was determined. Body lengths were measured from the tip of the nose along the ventral
midline to the base of the tail with the accuracy of 1 mm. Blood samples were obtained with cardiac punctures with sterile needles and syringes into test tubes containing EDTA and centrifuged at 4000g to obtain plasma. The livers, kidneys and testes were dissected. The samples were frozen in liquid nitrogen and stored at 40 C. 2.2. Hormone assays Plasma hormone concentrations were measured using radioimmunoassay (RIA) and enzyme-linked immunoassay (ELISA) methods. For the measurement of the testicular testosterone concentrations, one testis of each adult or juvenile male was homogenized in Dulbecco’s PBS buffer in ice (NaCl 8.0 g l1, KCl 0.2 g l1, Na2HPO4 1.15 g l1, KH2PO4 0.2 g l1, MgCl2 6 H2O 0.1 g l1, CaCl2 2 H2O 0.1 g l1 at pH 7.3–7.4). The liquid fraction was extracted with ether, vaporized in nitrogen and reconstituted into 0.1% BSA and used for RIA. The plasma tetraiodothyronine (T4), triiodothyronine (T3), testosterone, and progesterone concentrations were measured using the Spectria [125I] Coated Tube Radioimmunoassay kits (Orion Diagnostica, Espoo, Finland). The progesterone concentrations were measured only from the females that had born litters. The plasma leptin levels were measured with the Multi-Species Leptin RIA kit (Linco Research, St Charles, MO, USA) and the plasma ghrelin levels with the Ghrelin (Human) RIA kit (Phoenix Pharmaceuticals, Belmont, CA, USA). The plasma estradiol concentrations were determined using the immunoassay method (17b-Estradiol Immunoassay, R&D Systems, Wiesbaden-Nordenstadt, Germany). The PS mixture used in this experiment at concentrations up to 100 mg ml1 did not produce a positive OD reading in the used ELISA assay for estradiol. Some of the assays have been also previously used to measure hormone concentrations of the tundra vole (Mustonen et al., 2002) and other microtine (M. agrestis; Nieminen et al., 2002b, 2003) plasma. The assays were, however, validated such that serial dilutions of the tundra vole plasma showed linear changes that were parallel with the standard curve produced with the standards of the manufacturers (data not shown). For the actual measurements a Gamma Counter (1480 Wizard, Wallac, Turku, Finland) was used for the RIAs and the Multiscan Ascent microplate reader (Labsystems, Helsinki, Finland) for the ELISA analysis. 2.3. Measurement of enzyme activities The different enzyme activities were determined spectrophotometrically. The liver and kidney samples were weighed and homogenized in cold citrate buffer in pH 6.5 for the glucose-6-phosphatase (G6Pase), in pH 6.1 for the glycogen phosphorylase and in cold 0.85% sodium chloride for the lipase measurements. The activity
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of G6Pase was measured using glucose-6-phosphate as substrate in the presence of EDTA after an incubation time of 30 min at 37.5 C (Hers and van Hoof, 1966). The glycogen phosphorylase activity was measured in the presence of glucose-1-phosphate, glycogen, sodium fluoride and AMP according to the method of Hers and van Hoof (1966). The lipase activity was measured according to the method of Seligman and Nachlas (1962) using 2-naphthyl-laurate without taurocholate as substrate. Glycogen concentrations in the liver and kidney were measured spectrophotometrically according to the method of Lo et al. (1970). 2.4. Statistical analyses The data were analyzed with the two-way analysis of variance (ANOVA) with sex and PS treatment as determinants and using the post hoc Duncan’s test. Reproductive data (reproduced pairs in the control group vs. reproduced pairs in the PS group) were compared using the w2 test. The normality of distribution and the homogeneity of variances were determined with the Kolmogorov–Smirnov test and with the Levene test. Paired comparisons were performed with the Student’s t-test. The P < 0.05 level was considered to be statistically significant. The correlations were calculated using the Spearman’s correlation coefficient (rs) significant at P < 0.05 level. The results are expressed as mean SE. If there was no sexual dimorphism within a variable the results of the females and the males are presented together.
3. Results In adult females, the absolute cumulative food intake was higher in the PS treated voles than in the controls (two-way ANOVA, P < 0.05; Table 1), but PS had no effects on the BMs of the animals nor on their BM gain during the study period. Also the lengths of the animals and their absolute and relative organ weights were unaffected by the PS treatment. There were no observable effects caused by PS in any of these variables in the offspring. PS did not cause any effects on the age of the voles at reproduction or the time interval between mating and birth of offspring (Table 2). On the contrary, the percentage of successfully reproduces vole pairs was higher in the PS-treated group (Fig. 1, Table 3; w2 test, P < 0.001). Litter size or sex ratio were not affected by PS (Table 3). Nor was there any effect on the percentage of pairs that were barren or lost their entire litters during the experimental period. There was a significant negative correlation between the plasma estradiol concentrations and age of the parents at the birth of their offspring (rs=0.442, P< 0.05) and between the
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estradiol concentration and the time interval between mating and birth (rs=0.434, P < 0.05). The plasma and testicular testosterone concentrations of the PS-treated adult males were lower than in their controls (two-way ANOVA, P < 0.05; Table 4). The plasma and testicular testosterone and plasma estradiol concentrations of the reproduced and barren voles did not differ from each other, and due this, they were analysed together. The plasma testosterone concentrations correlated positively with the plasma T4 concentrations (rs=0.241, P< 0.05) and the estradiol concentrations with the plasma T3 levels (rs=0.419, P< 0.01). The plasma thyroid hormone, leptin or ghrelin concentrations were not influenced by PS treatment. However, the plasma leptin and ghrelin concentrations correlated negatively with each other (rs=0.265, P < 0.05) and the plasma ghrelin levels correlated inversely with the cumulative BM change (rs=0.337, P < 0.05) and with the plasma T3 concentrations (rs=0.367, P < 0.05), and the plasma leptin concentrations correlated positively with the plasma estradiol concentrations (rs=0.254, P< 0.05). In the offspring, the testicular testosterone concentrations were higher in the PS group, but no effect was observed in the plasma sex steroid levels. The liver and kidney enzyme activities were mostly unaffected by PS with the exception of the liver lipase activity that was higher in the PS-treated female offspring than in their controls (two-way ANOVA, P < 0.05; Table 4). There was, however, some sexual dimorphism in the results with higher liver glycogen content in the females than in the males (Table 4), but a higher kidney glycogen content in the males. The juvenile voles had higher liver and kidney G6Pase activities than adult voles, but their liver glycogen content was lower. The liver G6Pase activity correlated positively with the cumulative food intake of the voles (rs=0.375, P < 0.05).
4. Discussion PS have become a significant class of natural compounds in ecotoxicology and human risk assessment. The ecotoxicological risk of PS is hard to define as the principal source of PS in nature—pulp mill effluents— contain a mixture of PS and other organic compounds. This can lead to confusion about the actual active substances causing the endocrine disruption observed in fish or aquatic reptiles (Kovacs et al., 1997). Laboratory experiments in vitro or in vivo with a purified compound can offer more precise data as evidenced by several studies. In albino rats, subcutaneous PS causes a decrease in testicular weight and sperm count at 0.5–5 mg PS kg1 day1 (Moghadasian, 2000) and b-sitosterol per se inhibits the progesterone-induced acrosome reaction of human sperm (Khorasani et al., 2000). On the other
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Table 1 BM, body length, food intake and absolute and relative organ weights of adult and juvenile tundra voles according to PS exposure. Data are meanSE Adults Control males N=10
Juveniles Control females N=10
PS males N=13
PS females N=13
BM g begin of study 21.70.6ab 20.70.9a 25.11.1b 21.00.4a BM g end of study 33.32.0bc 31.31.7b 36.61.5c 32.71.3bc BM g 11.72.0 10.51.8 11.41.2 11.61.3 Body length cm 11.30.2b 11.00.1b 11.10.1b 10.90.1b Cumulative food intake g 0–14 wk 140.98.2b 117.44.3a 147.88.7b 135.75.3b Liver weight mg 118060b 1259136b 119563b 128895b Liver weight BM1 % 35.60.9 39.52.3 32.81.3 39.22.0 Kidney weight mg 33518bc 28422b 37314c 33322bc Kidney weight BM1 % 10.10.3a 9.20.6a 10.30.4a 10.30.5a Testicular weight mg 20120b 23811b Testicular weight BM1 % 6.00.5b 6.50.3b
Control males N=5
Control females N=7
PS males N=11
PS females N=9
14.60.3a 15.10.7a 11.90.34a 8.40.1a 8.50.1a
15.40.6a 16.00.9a 12.60.49a 8.40.1a 8.30.2a
55339a 38.11.6 1903a 13.10.3b 185a 1.20.3a
58725a 38.41.3 2037a 13.30.5b 183a 1.20.2a
53539a 35.32.8 2019a 13.20.4b
58934a 36.80.5 21110a 13.30.4b
According to Duncan’s test the means with no common letter differ at P<0.05 level (two-way ANOVA). a As the sex of young offspring could not be determined the BM change is the combined mean of all male and female offspring.
Table 2 Reproductive variables of reproduced adult tundra voles (19 pairs out of 23 mated pairs) according to PS exposure. Data are mean SE
Duration of the experiment (day) Age (day) beginning of study Mating age (day) Time interval between mating and birth Age (day) at birth of offspring Age (day) end of study
Control males N=10
Control females N=10
PS males N=13
PS females N=13
871 956 1256 384 1626 1825
916 964 122 5 414 163 7 187 8
87 7 93 2 126 6 32 3 158 6 180 7
813 941 118 3 344 152 5 175 6
hand, the beneficial effects of PS on the serum lipid profile are undisputed (Drexel et al., 1981; Miettinen et al., 1995; Mattson et al., 1982; Vanhanen et al., 1993). In this study, the PS mixture used was extracted from pulp mill effluents making it suitable when studying the possible risks of PS to natural ecosystems. The results of this study showed no significant mortality or morbidity in the PS-treated voles compared to the control animals. This supports the recent results of Kim et al. (2003), who have demonstrated that the noobserved-adverse-effect level of PS in the haematology or serum biochemistry of rats would be as high as 9000 mg PS kg1 day1. In this context, it is understandable that even the potentially more susceptible voles (Nieminen et al., 2003) did not show any adverse effects due to PS administration. It has been previously observed that the food intake of rats and voles increases with dietary PS or phytostanol supplement (Whittaker et al., 1999; Nieminen et al., 2003). The results of the present study support this with an increase in the cumulative food consumption of female voles. This has been attributed to a compensatory mechanism of the animals caused by a reduced intake of calories with an inhibition of cholesterol absorption in the intestine due to PS
(Jones et al., 2000). A similar effect could be the observed increase in the liver lipase activity in the female offspring due to PS treatment. PS interfere with cholesterol absorption in the gut (Moghadasian, 2000) and a more active lipid turnover could be required as a result of this. These results are unfortunately complicated by the fact
Fig. 1. The percentage of reproduced (voles that gave birth including pairs who lost entire litters) tundra vole pairs treated with PS supplement or the regular diet. ***=Significant difference between the PS and control pairs (w2 test, P <0.001).
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P. Nieminen et al. / Food and Chemical Toxicology 42 (2004) 945–951 Table 3 Reproductive variables of the tundra vole pairs according to PS exposure. Data are mean SE
N Reproduced N /% Barren N /% Sex ratio of offspring males/females% Gave birth but lost their entire litters N /% N of offspring/Mean litter size at the age of one day Surviving offspring at the age of 1 day N/offspring per pair Surviving offspring at the age of 1 wk N/offspring per pair Surviving offspring at the age of 2 wk N/offspring per pair Surviving offspring at the age of 3 wk N/offspring per pair Mean litter size at the age of 3 wk/per pair BM of whole litter/BM of an offspring at the age of 1 day BM of whole litter/BM of an offspring at the age of 1 wk BM of whole litter/BM of an offspring at the age of 2 wk a b
Control pairs
PS treated pairs
10 6/60%a 4/40%a 33/66 2/20% 17/2.80.5 16/1.6 13/1.3b 13/1.3 12/1.2 1.30.8 7.81.4/2.90.1 23.62.6/7.70.6 37.93.6/12.60.5
13 11/85%a 2/15%a 55/44 2/15% 38/3.50.3 33/3 21/1.9b 21/1.9 20/ 1.8 1.90.6 8.60.5/2.90.1 18.92.8/6.80.5 38.42.7/12.80.4
Significant difference between experimental groups (w2 test, P<0.001). Significant reduction in the number of surviving offspring from birth to 1 wk of age (paired sample t-test, P <0.01).
Table 4 Plasma (P) and testicular (T) hormone concentrations, liver (L) and kidney (K) glycogen content and enzyme activities of adult and juvenile voles according to PS exposure. Data are meanSE. G6Pase=glucose-6-phosphatase, yonly reproduced females included. nd=not determined Adults
P testosterone nmol l1 T testosterone ng mg1 P estradiol pmol l1 P progesterone nmol l1y P T4 nmol l1 P T3 nmol l1 P T3 T1 4 % P leptin ng ml1 P ghrelin ng ml1 L glycogen mg g1 K glycogen mg g1 L G6Pase mg P mg1 h1 K G6Pase mg P mg1 h1 L phosphorylase mg P mg1 h1 K phosphorylase mg P mg1 h1 L lipase mg 2-naphthol mg1 h1 K lipase mg 2-naphthol mg1 h1
Juveniles
Control males N=10
Control females N=10
0.83 0.27b 47.3 13.2c 524 66b nd 30.4 3.3ab 1.67 0.13 5.9 0.8 1.7 0.5 2.8 0.2 6.7 0.9a 0.70 0.06b 61.5 3.4ab 38.4 2.2a 22.8 2.2bc 4.4 0.3a 36.2 2.9a 26.9 1.8c
0.05 0.01a
PS males N=13
0.320.06a 28.83.0b 528 102b 55564b 47.7 22.0a nd 22.0 2.4a 32.52.6b 1.37 0.07 1.680.09 6.9 0.8 5.30.3 0.6 0.2 2.10.6 3.2 0.4 2.70.2 16.0 4.0b 9.52.3ab 0.45 0.07a 0.750.05b 58.0 4.3a 63.23.4ab 39.3 1.5a 36.70.9a 20.9 2.2abc 22.61.2bc 4.7 0.4ab 4.40.2a 40.6 2.2a 38.42.4a 23.7 2.3abc 27.70.9c
PS females N=13
Control males N=5
0.030.01a 0.050.02a 1.71.0aa 51758b 11736a 57.914.3a nd 22.51.8ab 30.67.4ab 1.470.11 nd 7.20.7 nd 1.80.5 1.20.5 2.50.2 2.50.2 16.42.4b 5.61.6a 0.460.06a 0.640.03ab 58.82.5ab 68.57.9abc 38.61.2a 45.31.6b 25.11.5c 15.80.7a 4.60.2ab 5.30.2abc 38.41.5a 57.55.0bc 24.81.6bc 18.82.8a
Control females N=7
PS males N=11
0.030.01a 0.020.01a 6.52.0aba 12637a 14219a nd nd 28.22.9ab 27.72.8ab nd nd nd nd 1.30.3 1.10.3 2.90.1 2.90.2 5.01.7a 2.90.8a 0.670.04b 0.610.02ab 76.74.2c 71.72.8bc 47.51.9bc 49.72.1bc 16.01.2a 17.31.2ab 6.20.5c 5.60.3bc 56.33.9b 61.72.7bc 20.80.9ab 21.31.2ab
PS females N=9 0.04 0.01a 84 8a nd 32.3 3.3b nd nd 0.7 0.2 2.6 0.4 3.3 0.6a 0.70 0.04b 69.9 3.2abc 51.5 1.5b 16.6 0.7a 5.3 0.4abc 66.1 4.2c 21.2 0.7ab
According to Duncan’s test the means with no common letter differ at P<0.05 (two-way ANOVA). a Difference between PS and control groups (t-test, P <0.02).
that during the mating period the measured food consumption represented the combined food intake of a male and a female vole. Thus the results on the food intake will need confirmation in future studies. There was a strong association between PS supplement and the percentage of reproducing vole pairs. This increase in the probability of reproduction by PS has to our knowledge not been described previously. However, no adverse effects on reproduction have been observed with plant stanol esters (Whittaker et al., 1999) or phytosterol esters at 1.54–5.62 g kg1 day1 (Waalkens-
Berendsen et al., 1999) in two-generation studies in rats. Plant stanols, of course, are absorbed at a much lower level in the intestine than PS are (Heinemann et al., 1993) and for this reason the effects of PS cannot be totally compared to the effects of stanols. However, in this study the effects of PS were clearly stronger at lower doses than the no-observed-adverse-effect level of rats emphasising the different responses of different rather closely related rodent species and the need of many different animal models in ecotoxicological risk assessment.
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Previous studies indicate that the average fertility index of tundra vole females of the North-American subspecies M. o. macfarlani is 70% (Dieterich and Preston, 1977; Morrison et al., 1976). Our control voles reproduced at a lower rate (60%) than this, but the PS treated voles had a fertility index (85%) above these previous findings. Litter size, sex ratio, the percentage of offspring surviving until weaning age, or most of the endocrine or enzymatic variables of young voles were unaffected. This suggests that the risks of parental PS consumption on reproduction are slight in voles, at least in the F1 generation. Of course, as a herbivore the tundra vole encounters diverse plant material in its diet and has consumed various PS throughout its evolutionary past. In fact, the increased reproduction in the PS group compared to the control voles or previous studies (Dieterich and Preston, 1977; Morrison et al., 1976) could indicate to an opposite effect. PS are compounds occurring in the natural foodstuffs of voles and the species encounters them constantly as a part of its natural diet. PS can offer the voles a suitable and constantly accessible store of steroids that could be remodelled into sex steroids (see also Moghadasian, 2000). As no exact data of the composition of previous diets of laboratory-raised tundra voles exist (Dieterich and Preston, 1977; Morrison et al., 1976) the precise effects of diet on the reproduction of this species require further studies. Also the relatively small number of pairs in this study makes the results preliminary and they will have to be confirmed in studies with a larger number of breeding voles. This strong association between the number of reproduced pairs and PS is puzzling, as the plasma and testicular testosterone concentrations were in fact significantly lower in the PS exposed male voles 50–60 days after mating. In previous studies with acute or subacute PS exposures, the plasma testosterone concentrations of voles have shown an increase at 5 mg PS kg1 day1 but no effect at a higher dose, 50 mg PS kg1 day1 (Nieminen et al., 2003). In a carnivore, the European polecat, there also seems to be a positive correlation between PS dose and plasma testosterone levels (Nieminen et al., 2002a). However, in fish exposed to b-sitosterol decreased sex steroid levels have been observed (Van der Kraak et al., 1992) indicating that altered circulating testosterone levels could be a common target of PS shared by these different vertebrates. Furthermore, dietary PS supplement of parents caused an increase in the testicular testosterone concentration of their male offspring possibly due to steroid synthesis from PS precursors (Moghadasian, 2000), or by other mechanisms during gestation or lactation. This effect has not been observed in humans after a PS supplement of 1.6 g day1 (Hendriks et al., 2003). However, one year is quite a short period in the human life cycle making it difficult to compare the results of this study to previous results on human exposure.
PS did not affect the other hormonal variables measured. The plasma ghrelin and leptin concentrations revealed, however, some interesting data on the functions of these hormones in wild rodents. In rats, ghrelin and leptin are antagonistic in the hypothalamic neuropeptide Y pathway (Shintani et al., 2001). This supports the results of this study with the negative correlation between the plasma leptin and ghrelin concentrations of the voles with similar mean values as in a previous tundra vole experiment (Mustonen et al., 2002). The observed negative correlation between the plasma ghrelin levels and the cumulative food intake of the voles, however, is more enigmatic. Ghrelin is considered to induce adiposity (Tscho¨p et al., 2000), which would be observed as a positive correlation between these variables. No specific mechanism behind this phenomenon is evident in the results of this study and further experiments will be needed to explain it. The observed positive correlation between leptin and estradiol suggests that leptin could be involved also in tundra vole reproduction. This is supported by human findings with a positive correlation between plasma leptin and estradiol concentrations during and after pregnancy (Hardie et al., 1997). For ecotoxicological risk assessment it is crucial to investigate the effects of PS also in wild mammals with more susceptibility to the effects of endocrine disruptors than laboratory rodents. Our results implicate that the effects of PS are widespread. PS do not only affect circulating cholesterol concentrations or attach to steroid receptors. Instead, there are complex interactions between physiological systems requiring further studies. In previous experiments no effects of phytostanols or PS have been observed in laboratory rat reproduction. The increased probability of reproduction and decreased testesterone concentrations of male voles observed in this preliminary study could have potential for monitoring the possible effects of PS in natural ecosystems by comparing exposed populations to populations inhabiting pristine environments without e.g. pulp mill effluent discharge.
Acknowledgements We sincerely thank Mrs. Anita Kervinen and Mr. Ari Ryo¨kkynen for their invaluable help in the analyses. Mr. Ulf Hotanen provided us with the PS mixture. This work was financially supported by the Academy of Finland and by the Maj and Tor Nessling Foundation.
References Baker, V.A., Hepburn, P.A., Kennedy, S.J., Jones, P.A., Lea, L.J., Sumpter, J.P., Ashby, J., 1999. Safety evaluation of phytosterol esters. Part 1. Assessment and oestrogenicity using a combination of in vivo and in vitro assays. Food and Chemical Toxicology 37, 13– 22.
P. Nieminen et al. / Food and Chemical Toxicology 42 (2004) 945–951 Dieterich, R.A., Preston, D.J., 1977. The tundra vole (Microtus oeconomus) as a laboratory animal. Laboratory Animal Science 27, 500–506. Drexel, H., Breier, C., Lisch, H.-J., Sailer, S., 1981. Lowering plasma cholesterol with beta-sitosterol and diet. Lancet 23, 1157. Hallikainen, M.A., Sarkkinen, E.S., Gylling, H., Erkkila¨, A.T., Uusitupa, M.I.J., 2000. Comparison of the effects of plant sterol ester and plant stanol ester-enriched margarines in lowering serum cholesterol concentrations in hypercholesterolaemic subjects on a low-fat diet. European Journal of Clinical Nutrition 54, 715–725. Hardie, L., Trayhurn, P., Abramovich, D., Fowler, P., 1997. Circulating leptin in women: a longitudinal study in the menstrual cycle and during pregnancy. Clinical Endocrinology 47, 101–106. Heinemann, T., Axtmann, G., von Bergmann, K., 1993. Comparison of intestinal absorption of cholesterol with different plant sterols in man. European Journal of Clinical Investigation 23, 827–831. Hendriks, H.F., Brink, E.J., Meijer, G.W., Princen, H.M., Ntanios, F.Y., 2003. Safety of long-term consumption of plant sterol estersenriched spread. European Journal of Clinical Nutrition 57, 681–692. Hers, H.G., van Hoof, F., 1966. Enzymes of glycogen degradation in biopsy material. In: Colowick, S., Kaplan, N.O. (Eds.), Methods in Enzymology. Vol. VIII. Academic Press, New York, pp. 525–532. Jones, P.J., Raeini-Sarjaz, M., Ntanios, F.Y., Vanstone, C.A., Feng, J.Y., Parsons, W.E., 2000. Modulation of plasma lipid levels and cholesterol kinetics by phytosterol versus phytostanol esters. Journal of Lipid Research 41, 697–705. Khorasani, A.M., Cheung, A.P., Lee, C.Y.G., 2000. Cholesterol inhibitory effects on human sperm-induced acrosome reaction. Journal of Andrology 21, 586–594. Kim, J.-C., Kang, B.-H., Shin, C.-C., Kim, Y.-B., Lee, H.-S., Kim, C.Y., Han, J., Kim, K.-S., Chung, D.-W., Chung, M.-K., 2003. Subchronic toxicity of plant sterol esters administered by gavage to Sprague-Dawley rats. Food and Chemical Toxicology 40, 1569–1580. Kovacs, T.G., Voss, R.H., Megraw, S.R., Martel, P.H., 1997. Perspectives on Canadian field studies examining the potential of pulp and paper mill effluent to affect fish reproduction. Journal of Toxicology and Environmental Health 51, 305–352. Lo, S., Russell, J.C., Taylor, A.W., 1970. Determination of glycogen in small tissue samples. Journal of Applied Physiology 28, 234–236. Mattson, F.H., Grundy, S.M., Crouse, J., 1982. Optimizing the effect of plant sterols on cholesterol absorption in man. Americal Journal of Clinical Nutrition 35, 697–700. Mellanen, P., Peta¨nen, T., Lehtima¨ki, J., Ma¨kela¨, S., Bylynd, G., Holmbom, B., Mannila, E., Oikari, A., Santti, R., 1996. Woodderived estrogens: studies in vitro with breast cancer cell lines and in vivo in trout. Toxicology and Applied Pharmacology 136, 381–388. Miettinen, T.A., Puska, P., Gylling, H., Vanhanen, H., Vartiainen, E., 1995. Serum cholesterol lowering by sitostanol ester margarine in a mildly hypercholesterolemic random population. New England Journal of Medicine 333, 1308–1312. Moghadasian, M.H., 2000. Pharmacological properties of plant sterols. In vivo and in vitro observations. Life Sciences 67, 605–615.
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Morrison, P., Dieterich, R., Preston, D., 1976. Breeding and reproduction of fifteen wild rodents maintained as laboratory colonies. Laboratory Animal Science 26, 237–243. Mustonen, A.M., Nieminen, P., Hyva¨rinen, H., 2002. Melatonin and the wintering strategy of the tundra vole, Microtus oeconomus. Zoological Science 19, 683–687. Nieminen, P., Mustonen, A.-M., Lindstro¨m-Seppa¨, P., Asikainen, J., Mussalo-Rauhamaa, H., Kukkonen, J.V.K., 2002a. Phytosterols act as endocrine and metabolic disruptors in the European polecat (Mustela putorius). Toxicology and Applied Pharmacology 178, 22–28. Nieminen, P., Lindstro¨m-Seppa¨, P., Mustonen, A.-M., MussaloRauhamaa, H., Kukkonen, J.V.K., 2002b. Bisphenol A affects endocrine physiology and biotransformation enzyme activities of the field vole (Microtus agrestis). General and Comparative Endocrinology 126, 183–189. Nieminen, P., Mustonen, A.-M., Lindstro¨m-Seppa¨, P., Ka¨rkka¨inen, V., Mussalo-Rauhamaa, H., Kukkonen, J.V.K., 2003. Phytosterols affect endocrinology and metabolism of the field vole (Microtus agrestis). Experimental Biology and Medicine 228, 188–193. Seligman, A.M., Nachlas, M.M., 1962. Lipase. In: Bermayer, H.U. (Ed.), Methoden der Enzymatischen Analyse. Verlag Chemie GmbH, Weinheim, pp. 776–778. Shintani, M., Ogawa, Y., Ebihara, K., Aizawa-Abe, M., Miyanaga, F., Takaya, K., Hayashi, T., Inoue, G., Hosoda, K., Kojima, M., Kangawa, K., Nakao, K., 2001. Ghrelin, an endogenous growth hormone secretagogue, is a novel orexigenic peptide that antagonizes leptin action through the activation of hypothalamic neuropeptide Y/Y1 receptor pathway. Diabetes 50, 227–232. Tast, J., 1966. The root vole, Microtus oeconomus (Pallas), as an inhabitant of seasonally flooded land. Annales Zoologici Fennici 3, 127–171. Tscho¨p, M., Smiley, D.L., Heiman, M.L., 2000. Ghrelin induces adiposity in rodents. Nature 407, 908–913. Van der Kraak, G.J., Munkittrick, K.R., McMaster, M.E., Portt, C.B., Chang, J.P., 1992. Exposure to bleached kraft pulp mill effluent disrupts the pituitary-gonadal axis of white sucker at multiple sites. Toxicology and Applied Pharmacology 115, 224–233. Vanhanen, H.T., Blomquist, S., Enholm, C., Hyvo¨nen, M., Jauhiainen, M., Torstila, I., Miettinen, T.A., 1993. Serum cholesterol, cholesterol precursors, and plant sterols in hypercholesterolemic subjects with different apoE phenotypes during dietary sitostanol ester treatment. Journal of Lipid Research 34, 1535–1544. Waalkens-Berendsen, D.H., Wolterbeek, A.P.M., Wijnands, M.V.W., Richold, M., Hepburn, P.A., 1999. Safety evaluation of phytosterol esters. Part 3. Two-generation reproduction study in rats with phytosterol esters—a novel functional food. Food and Chemical Toxicology 37, 683–696. Whittaker, M.H., Frankos, V.H., Wolterbeek, A.P.M., WaalkensBerendsen, D.H., 1999. Two-generation reproductive toxicity study of plant stanol esters in rats. Regulatory Toxicology and Pharmacology 29, 196–204.
Reproduction of the tundra vole (Microtus oeconomus) with dietary phytosterol supplement P. Nieminen*, A.-M. Mustonen, P. Pa¨iva¨la¨inen, J.V.K. Kukkonen University of Joensuu, Department of Biology, PO Box 111, FIN-80101 Joensuu, Finland Received 6 August 2003; accepted 7 February 2004
Abstract Phytosterols (PS) are plant-derived compounds with estrogenic activity in vitro and beneficial effects on the serum lipid profile in vivo. In nature, PS exposure can derive from pulp mill effluents. The effects of a pulp-mill derived PS mixture on the reproduction, endocrine variables and enzyme activities of the tundra vole (Microtus oeconomus) were investigated in a two-generation study. The cumulative food intake of PS-treated females was higher than in the control group supporting previous results on the effects of PS on food consumption in rodents. 85% of the PS treated pairs reproduced, but the figure was only 60% for the control pairs. The plasma and testicular testosterone concentrations were lower in the adult PS males, but the PS-treated male offspring had higher testicular testosterone concentrations than their controls. In the female offspring, the liver lipase activity was higher in the PS-treated group, which could be a result of decreased cholesterol absorption in the gut. Chronic PS treatment increased the reproduction probability of the species and had a potential effect on the sex steroid hormones of maturing offspring, which could have applications in environmental monitoring. # 2004 Elsevier Ltd. All rights reserved. Keywords: Microtus oeconomus; Reproduction; Sitosterol; Testosterone; Tundra vole
1. Introduction The beneficial effects of phytosterols (PS) and phytostanols to lower elevated total and low density lipoprotein cholesterol levels have been thoroughly documented (Drexel et al., 1981; Miettinen et al., 1995; Jones et al., 2000). Due to this, PS are added to margarines at 30 mg kg1 day1 (Hallikainen et al., 2000). Pulp mill effluents are a source of PS in nature. PS cause reduced sex steroid levels and gonad size in fish (Van der Kraak et al., 1992; Mellanen et al., 1996). b-Sitosterol induces vitellogenin gene expression in the male rainbow trout (Oncorhynchus mykiss) and it also has estrogenic activity in T-47D breast cancer cells (Mellanen et al., 1996). In rats, subcutaneous b-sitosterol at doses of 0.5–5 mg kg1 day1 reduces sperm count and testicular weight (Moghadasian, 2000). However, PS mixtures do not cause estrogenicity at 0–500 mg kg1 day1 measured with effects on uterine weight of immature female rats * Corresponding author. Tel.: +358-13251-3576; fax: +358-132513590. E-mail address: [email protected].fi (P. Nieminen). 0278-6915/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2004.02.006
(Baker et al., 1999). In European polecats (Mustela putorius), PS exposure leads to increases in the circulating estradiol and thyroid hormone levels (Nieminen et al., 2002a). This may be caused by PS being used as precursors of sex steroids in gonads (Moghadasian, 2000). The tundra vole (Microtus oeconomus, Pallas, 1776) is a small rodent with a circumpolar distribution (Tast, 1966). Its diet consists of diverse leaves, flowers, seeds and stalks of different grass or sedge species. The species is a seasonal long-day breeder and reproduction can be induced in laboratories by exposing the animals to long daylength. Previously it has been documented in dose– response studies on microtines that a two-week exposure to PS at 5 mg kg1 day1 increases the circulating testosterone concentrations of male voles and the liver glycogen phosphorylase activities of both sexes (Nieminen et al., 2003). As a regionally abundant circumpolar species the tundra vole also has potential as a bioindicator of possible endocrine disruption in arctic and boreal nature. The aim of the present study was to investigate the effects of PS on the reproduction, endocrinology and parameters of intermediary metabolism of adult voles and on the physiology of their F1 offspring.
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2. Materials and methods 2.1. Animals and exposure Forty-six tundra voles (Microtus oeconomus; 23 males, 23 females, age 91 6 days; sexual maturity reached at the age of 70–80 days) from the breeding colony of the University of Joensuu were selected for the study. The animals were housed singly in solid bottom plastic cages (Makrolon: 422215 cm) with wood shavings for bedding and free access to water and a pelleted diet (Avelsfoder fo¨r ra˚tta och mus R36: protein 18.5%, fat 4.0%, energy content 1260 kJ 100 g1, Lactamin, Stockholm, Sweden). The animals were assigned randomly into two experimental groups. The control group (10 pairs) receiving the regular diet and the PS group (13 pairs). Originally, the number of pairs in both experimental groups was the same (14 control and 14 PS treated pairs). Due to difficulties in the determination of sex of juvenile voles some of the formed pairs were female– female pairs and these were excluded from the analyses. The PS group received peroral PS (Ultra-sitosterol1: 88.7% b-sitosterol+b-sitostanol, 9.0% campesterol +campestanol, 0.9% artenols; mixture derived from pulp mill effluents by UPM Kymmene, Kaukas, Lappeenranta, Finland) mixed into the normal food of the animals at 5 mg kg1 day1. This is a single dose study; other doses were not needed as the dose is based on previous dose-response studies showing significant effects on microtine voles at this fairly low dose of PS, such as increased plasma testosterone and estradiol concentrations in male voles with an opposite trend of plasma luteinizing hormone concentrations (Nieminen et al., 2003). At a higher dose (50 mg kg1 day1) there was a return to the level of control animals. Yet 5 mg kg1 day1 is a relevant dose, as it is the recommended dose of PS for several natural remedies and can be also encountered in nature. After four weeks, the animals were paired, allowed to mate and reproduce freely. After parturition, the male was removed from the cage. Food intake was measured weekly by weighing the amount of food left uneaten by an individual vole or a pair at the end of each study week. Food intake during mating period represents the average of the male–female pair and during the lactating period mostly that of the lactating female, but closer to the weaning age of three weeks, the young voles also consumed some of the pellets. The body mass (BM) of all the animals was measured weekly, and the BMs of the offspring at the age of 1, 7 and 14 days. The Animals Care and Use Committee of the University of Joensuu approved all procedures. Both the parents and the offspring were sacrificed three weeks after parturition (weaning age of the offspring). The animals were euthanized quickly with diethyl ether and their BM was determined. Body lengths were measured from the tip of the nose along the ventral
midline to the base of the tail with the accuracy of 1 mm. Blood samples were obtained with cardiac punctures with sterile needles and syringes into test tubes containing EDTA and centrifuged at 4000g to obtain plasma. The livers, kidneys and testes were dissected. The samples were frozen in liquid nitrogen and stored at 40 C. 2.2. Hormone assays Plasma hormone concentrations were measured using radioimmunoassay (RIA) and enzyme-linked immunoassay (ELISA) methods. For the measurement of the testicular testosterone concentrations, one testis of each adult or juvenile male was homogenized in Dulbecco’s PBS buffer in ice (NaCl 8.0 g l1, KCl 0.2 g l1, Na2HPO4 1.15 g l1, KH2PO4 0.2 g l1, MgCl2 6 H2O 0.1 g l1, CaCl2 2 H2O 0.1 g l1 at pH 7.3–7.4). The liquid fraction was extracted with ether, vaporized in nitrogen and reconstituted into 0.1% BSA and used for RIA. The plasma tetraiodothyronine (T4), triiodothyronine (T3), testosterone, and progesterone concentrations were measured using the Spectria [125I] Coated Tube Radioimmunoassay kits (Orion Diagnostica, Espoo, Finland). The progesterone concentrations were measured only from the females that had born litters. The plasma leptin levels were measured with the Multi-Species Leptin RIA kit (Linco Research, St Charles, MO, USA) and the plasma ghrelin levels with the Ghrelin (Human) RIA kit (Phoenix Pharmaceuticals, Belmont, CA, USA). The plasma estradiol concentrations were determined using the immunoassay method (17b-Estradiol Immunoassay, R&D Systems, Wiesbaden-Nordenstadt, Germany). The PS mixture used in this experiment at concentrations up to 100 mg ml1 did not produce a positive OD reading in the used ELISA assay for estradiol. Some of the assays have been also previously used to measure hormone concentrations of the tundra vole (Mustonen et al., 2002) and other microtine (M. agrestis; Nieminen et al., 2002b, 2003) plasma. The assays were, however, validated such that serial dilutions of the tundra vole plasma showed linear changes that were parallel with the standard curve produced with the standards of the manufacturers (data not shown). For the actual measurements a Gamma Counter (1480 Wizard, Wallac, Turku, Finland) was used for the RIAs and the Multiscan Ascent microplate reader (Labsystems, Helsinki, Finland) for the ELISA analysis. 2.3. Measurement of enzyme activities The different enzyme activities were determined spectrophotometrically. The liver and kidney samples were weighed and homogenized in cold citrate buffer in pH 6.5 for the glucose-6-phosphatase (G6Pase), in pH 6.1 for the glycogen phosphorylase and in cold 0.85% sodium chloride for the lipase measurements. The activity
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of G6Pase was measured using glucose-6-phosphate as substrate in the presence of EDTA after an incubation time of 30 min at 37.5 C (Hers and van Hoof, 1966). The glycogen phosphorylase activity was measured in the presence of glucose-1-phosphate, glycogen, sodium fluoride and AMP according to the method of Hers and van Hoof (1966). The lipase activity was measured according to the method of Seligman and Nachlas (1962) using 2-naphthyl-laurate without taurocholate as substrate. Glycogen concentrations in the liver and kidney were measured spectrophotometrically according to the method of Lo et al. (1970). 2.4. Statistical analyses The data were analyzed with the two-way analysis of variance (ANOVA) with sex and PS treatment as determinants and using the post hoc Duncan’s test. Reproductive data (reproduced pairs in the control group vs. reproduced pairs in the PS group) were compared using the w2 test. The normality of distribution and the homogeneity of variances were determined with the Kolmogorov–Smirnov test and with the Levene test. Paired comparisons were performed with the Student’s t-test. The P < 0.05 level was considered to be statistically significant. The correlations were calculated using the Spearman’s correlation coefficient (rs) significant at P < 0.05 level. The results are expressed as mean SE. If there was no sexual dimorphism within a variable the results of the females and the males are presented together.
3. Results In adult females, the absolute cumulative food intake was higher in the PS treated voles than in the controls (two-way ANOVA, P < 0.05; Table 1), but PS had no effects on the BMs of the animals nor on their BM gain during the study period. Also the lengths of the animals and their absolute and relative organ weights were unaffected by the PS treatment. There were no observable effects caused by PS in any of these variables in the offspring. PS did not cause any effects on the age of the voles at reproduction or the time interval between mating and birth of offspring (Table 2). On the contrary, the percentage of successfully reproduces vole pairs was higher in the PS-treated group (Fig. 1, Table 3; w2 test, P < 0.001). Litter size or sex ratio were not affected by PS (Table 3). Nor was there any effect on the percentage of pairs that were barren or lost their entire litters during the experimental period. There was a significant negative correlation between the plasma estradiol concentrations and age of the parents at the birth of their offspring (rs=0.442, P< 0.05) and between the
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estradiol concentration and the time interval between mating and birth (rs=0.434, P < 0.05). The plasma and testicular testosterone concentrations of the PS-treated adult males were lower than in their controls (two-way ANOVA, P < 0.05; Table 4). The plasma and testicular testosterone and plasma estradiol concentrations of the reproduced and barren voles did not differ from each other, and due this, they were analysed together. The plasma testosterone concentrations correlated positively with the plasma T4 concentrations (rs=0.241, P< 0.05) and the estradiol concentrations with the plasma T3 levels (rs=0.419, P< 0.01). The plasma thyroid hormone, leptin or ghrelin concentrations were not influenced by PS treatment. However, the plasma leptin and ghrelin concentrations correlated negatively with each other (rs=0.265, P < 0.05) and the plasma ghrelin levels correlated inversely with the cumulative BM change (rs=0.337, P < 0.05) and with the plasma T3 concentrations (rs=0.367, P < 0.05), and the plasma leptin concentrations correlated positively with the plasma estradiol concentrations (rs=0.254, P< 0.05). In the offspring, the testicular testosterone concentrations were higher in the PS group, but no effect was observed in the plasma sex steroid levels. The liver and kidney enzyme activities were mostly unaffected by PS with the exception of the liver lipase activity that was higher in the PS-treated female offspring than in their controls (two-way ANOVA, P < 0.05; Table 4). There was, however, some sexual dimorphism in the results with higher liver glycogen content in the females than in the males (Table 4), but a higher kidney glycogen content in the males. The juvenile voles had higher liver and kidney G6Pase activities than adult voles, but their liver glycogen content was lower. The liver G6Pase activity correlated positively with the cumulative food intake of the voles (rs=0.375, P < 0.05).
4. Discussion PS have become a significant class of natural compounds in ecotoxicology and human risk assessment. The ecotoxicological risk of PS is hard to define as the principal source of PS in nature—pulp mill effluents— contain a mixture of PS and other organic compounds. This can lead to confusion about the actual active substances causing the endocrine disruption observed in fish or aquatic reptiles (Kovacs et al., 1997). Laboratory experiments in vitro or in vivo with a purified compound can offer more precise data as evidenced by several studies. In albino rats, subcutaneous PS causes a decrease in testicular weight and sperm count at 0.5–5 mg PS kg1 day1 (Moghadasian, 2000) and b-sitosterol per se inhibits the progesterone-induced acrosome reaction of human sperm (Khorasani et al., 2000). On the other
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Table 1 BM, body length, food intake and absolute and relative organ weights of adult and juvenile tundra voles according to PS exposure. Data are meanSE Adults Control males N=10
Juveniles Control females N=10
PS males N=13
PS females N=13
BM g begin of study 21.70.6ab 20.70.9a 25.11.1b 21.00.4a BM g end of study 33.32.0bc 31.31.7b 36.61.5c 32.71.3bc BM g 11.72.0 10.51.8 11.41.2 11.61.3 Body length cm 11.30.2b 11.00.1b 11.10.1b 10.90.1b Cumulative food intake g 0–14 wk 140.98.2b 117.44.3a 147.88.7b 135.75.3b Liver weight mg 118060b 1259136b 119563b 128895b Liver weight BM1 % 35.60.9 39.52.3 32.81.3 39.22.0 Kidney weight mg 33518bc 28422b 37314c 33322bc Kidney weight BM1 % 10.10.3a 9.20.6a 10.30.4a 10.30.5a Testicular weight mg 20120b 23811b Testicular weight BM1 % 6.00.5b 6.50.3b
Control males N=5
Control females N=7
PS males N=11
PS females N=9
14.60.3a 15.10.7a 11.90.34a 8.40.1a 8.50.1a
15.40.6a 16.00.9a 12.60.49a 8.40.1a 8.30.2a
55339a 38.11.6 1903a 13.10.3b 185a 1.20.3a
58725a 38.41.3 2037a 13.30.5b 183a 1.20.2a
53539a 35.32.8 2019a 13.20.4b
58934a 36.80.5 21110a 13.30.4b
According to Duncan’s test the means with no common letter differ at P<0.05 level (two-way ANOVA). a As the sex of young offspring could not be determined the BM change is the combined mean of all male and female offspring.
Table 2 Reproductive variables of reproduced adult tundra voles (19 pairs out of 23 mated pairs) according to PS exposure. Data are mean SE
Duration of the experiment (day) Age (day) beginning of study Mating age (day) Time interval between mating and birth Age (day) at birth of offspring Age (day) end of study
Control males N=10
Control females N=10
PS males N=13
PS females N=13
871 956 1256 384 1626 1825
916 964 122 5 414 163 7 187 8
87 7 93 2 126 6 32 3 158 6 180 7
813 941 118 3 344 152 5 175 6
hand, the beneficial effects of PS on the serum lipid profile are undisputed (Drexel et al., 1981; Miettinen et al., 1995; Mattson et al., 1982; Vanhanen et al., 1993). In this study, the PS mixture used was extracted from pulp mill effluents making it suitable when studying the possible risks of PS to natural ecosystems. The results of this study showed no significant mortality or morbidity in the PS-treated voles compared to the control animals. This supports the recent results of Kim et al. (2003), who have demonstrated that the noobserved-adverse-effect level of PS in the haematology or serum biochemistry of rats would be as high as 9000 mg PS kg1 day1. In this context, it is understandable that even the potentially more susceptible voles (Nieminen et al., 2003) did not show any adverse effects due to PS administration. It has been previously observed that the food intake of rats and voles increases with dietary PS or phytostanol supplement (Whittaker et al., 1999; Nieminen et al., 2003). The results of the present study support this with an increase in the cumulative food consumption of female voles. This has been attributed to a compensatory mechanism of the animals caused by a reduced intake of calories with an inhibition of cholesterol absorption in the intestine due to PS
(Jones et al., 2000). A similar effect could be the observed increase in the liver lipase activity in the female offspring due to PS treatment. PS interfere with cholesterol absorption in the gut (Moghadasian, 2000) and a more active lipid turnover could be required as a result of this. These results are unfortunately complicated by the fact
Fig. 1. The percentage of reproduced (voles that gave birth including pairs who lost entire litters) tundra vole pairs treated with PS supplement or the regular diet. ***=Significant difference between the PS and control pairs (w2 test, P <0.001).
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P. Nieminen et al. / Food and Chemical Toxicology 42 (2004) 945–951 Table 3 Reproductive variables of the tundra vole pairs according to PS exposure. Data are mean SE
N Reproduced N /% Barren N /% Sex ratio of offspring males/females% Gave birth but lost their entire litters N /% N of offspring/Mean litter size at the age of one day Surviving offspring at the age of 1 day N/offspring per pair Surviving offspring at the age of 1 wk N/offspring per pair Surviving offspring at the age of 2 wk N/offspring per pair Surviving offspring at the age of 3 wk N/offspring per pair Mean litter size at the age of 3 wk/per pair BM of whole litter/BM of an offspring at the age of 1 day BM of whole litter/BM of an offspring at the age of 1 wk BM of whole litter/BM of an offspring at the age of 2 wk a b
Control pairs
PS treated pairs
10 6/60%a 4/40%a 33/66 2/20% 17/2.80.5 16/1.6 13/1.3b 13/1.3 12/1.2 1.30.8 7.81.4/2.90.1 23.62.6/7.70.6 37.93.6/12.60.5
13 11/85%a 2/15%a 55/44 2/15% 38/3.50.3 33/3 21/1.9b 21/1.9 20/ 1.8 1.90.6 8.60.5/2.90.1 18.92.8/6.80.5 38.42.7/12.80.4
Significant difference between experimental groups (w2 test, P<0.001). Significant reduction in the number of surviving offspring from birth to 1 wk of age (paired sample t-test, P <0.01).
Table 4 Plasma (P) and testicular (T) hormone concentrations, liver (L) and kidney (K) glycogen content and enzyme activities of adult and juvenile voles according to PS exposure. Data are meanSE. G6Pase=glucose-6-phosphatase, yonly reproduced females included. nd=not determined Adults
P testosterone nmol l1 T testosterone ng mg1 P estradiol pmol l1 P progesterone nmol l1y P T4 nmol l1 P T3 nmol l1 P T3 T1 4 % P leptin ng ml1 P ghrelin ng ml1 L glycogen mg g1 K glycogen mg g1 L G6Pase mg P mg1 h1 K G6Pase mg P mg1 h1 L phosphorylase mg P mg1 h1 K phosphorylase mg P mg1 h1 L lipase mg 2-naphthol mg1 h1 K lipase mg 2-naphthol mg1 h1
Juveniles
Control males N=10
Control females N=10
0.83 0.27b 47.3 13.2c 524 66b nd 30.4 3.3ab 1.67 0.13 5.9 0.8 1.7 0.5 2.8 0.2 6.7 0.9a 0.70 0.06b 61.5 3.4ab 38.4 2.2a 22.8 2.2bc 4.4 0.3a 36.2 2.9a 26.9 1.8c
0.05 0.01a
PS males N=13
0.320.06a 28.83.0b 528 102b 55564b 47.7 22.0a nd 22.0 2.4a 32.52.6b 1.37 0.07 1.680.09 6.9 0.8 5.30.3 0.6 0.2 2.10.6 3.2 0.4 2.70.2 16.0 4.0b 9.52.3ab 0.45 0.07a 0.750.05b 58.0 4.3a 63.23.4ab 39.3 1.5a 36.70.9a 20.9 2.2abc 22.61.2bc 4.7 0.4ab 4.40.2a 40.6 2.2a 38.42.4a 23.7 2.3abc 27.70.9c
PS females N=13
Control males N=5
0.030.01a 0.050.02a 1.71.0aa 51758b 11736a 57.914.3a nd 22.51.8ab 30.67.4ab 1.470.11 nd 7.20.7 nd 1.80.5 1.20.5 2.50.2 2.50.2 16.42.4b 5.61.6a 0.460.06a 0.640.03ab 58.82.5ab 68.57.9abc 38.61.2a 45.31.6b 25.11.5c 15.80.7a 4.60.2ab 5.30.2abc 38.41.5a 57.55.0bc 24.81.6bc 18.82.8a
Control females N=7
PS males N=11
0.030.01a 0.020.01a 6.52.0aba 12637a 14219a nd nd 28.22.9ab 27.72.8ab nd nd nd nd 1.30.3 1.10.3 2.90.1 2.90.2 5.01.7a 2.90.8a 0.670.04b 0.610.02ab 76.74.2c 71.72.8bc 47.51.9bc 49.72.1bc 16.01.2a 17.31.2ab 6.20.5c 5.60.3bc 56.33.9b 61.72.7bc 20.80.9ab 21.31.2ab
PS females N=9 0.04 0.01a 84 8a nd 32.3 3.3b nd nd 0.7 0.2 2.6 0.4 3.3 0.6a 0.70 0.04b 69.9 3.2abc 51.5 1.5b 16.6 0.7a 5.3 0.4abc 66.1 4.2c 21.2 0.7ab
According to Duncan’s test the means with no common letter differ at P<0.05 (two-way ANOVA). a Difference between PS and control groups (t-test, P <0.02).
that during the mating period the measured food consumption represented the combined food intake of a male and a female vole. Thus the results on the food intake will need confirmation in future studies. There was a strong association between PS supplement and the percentage of reproducing vole pairs. This increase in the probability of reproduction by PS has to our knowledge not been described previously. However, no adverse effects on reproduction have been observed with plant stanol esters (Whittaker et al., 1999) or phytosterol esters at 1.54–5.62 g kg1 day1 (Waalkens-
Berendsen et al., 1999) in two-generation studies in rats. Plant stanols, of course, are absorbed at a much lower level in the intestine than PS are (Heinemann et al., 1993) and for this reason the effects of PS cannot be totally compared to the effects of stanols. However, in this study the effects of PS were clearly stronger at lower doses than the no-observed-adverse-effect level of rats emphasising the different responses of different rather closely related rodent species and the need of many different animal models in ecotoxicological risk assessment.
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Previous studies indicate that the average fertility index of tundra vole females of the North-American subspecies M. o. macfarlani is 70% (Dieterich and Preston, 1977; Morrison et al., 1976). Our control voles reproduced at a lower rate (60%) than this, but the PS treated voles had a fertility index (85%) above these previous findings. Litter size, sex ratio, the percentage of offspring surviving until weaning age, or most of the endocrine or enzymatic variables of young voles were unaffected. This suggests that the risks of parental PS consumption on reproduction are slight in voles, at least in the F1 generation. Of course, as a herbivore the tundra vole encounters diverse plant material in its diet and has consumed various PS throughout its evolutionary past. In fact, the increased reproduction in the PS group compared to the control voles or previous studies (Dieterich and Preston, 1977; Morrison et al., 1976) could indicate to an opposite effect. PS are compounds occurring in the natural foodstuffs of voles and the species encounters them constantly as a part of its natural diet. PS can offer the voles a suitable and constantly accessible store of steroids that could be remodelled into sex steroids (see also Moghadasian, 2000). As no exact data of the composition of previous diets of laboratory-raised tundra voles exist (Dieterich and Preston, 1977; Morrison et al., 1976) the precise effects of diet on the reproduction of this species require further studies. Also the relatively small number of pairs in this study makes the results preliminary and they will have to be confirmed in studies with a larger number of breeding voles. This strong association between the number of reproduced pairs and PS is puzzling, as the plasma and testicular testosterone concentrations were in fact significantly lower in the PS exposed male voles 50–60 days after mating. In previous studies with acute or subacute PS exposures, the plasma testosterone concentrations of voles have shown an increase at 5 mg PS kg1 day1 but no effect at a higher dose, 50 mg PS kg1 day1 (Nieminen et al., 2003). In a carnivore, the European polecat, there also seems to be a positive correlation between PS dose and plasma testosterone levels (Nieminen et al., 2002a). However, in fish exposed to b-sitosterol decreased sex steroid levels have been observed (Van der Kraak et al., 1992) indicating that altered circulating testosterone levels could be a common target of PS shared by these different vertebrates. Furthermore, dietary PS supplement of parents caused an increase in the testicular testosterone concentration of their male offspring possibly due to steroid synthesis from PS precursors (Moghadasian, 2000), or by other mechanisms during gestation or lactation. This effect has not been observed in humans after a PS supplement of 1.6 g day1 (Hendriks et al., 2003). However, one year is quite a short period in the human life cycle making it difficult to compare the results of this study to previous results on human exposure.
PS did not affect the other hormonal variables measured. The plasma ghrelin and leptin concentrations revealed, however, some interesting data on the functions of these hormones in wild rodents. In rats, ghrelin and leptin are antagonistic in the hypothalamic neuropeptide Y pathway (Shintani et al., 2001). This supports the results of this study with the negative correlation between the plasma leptin and ghrelin concentrations of the voles with similar mean values as in a previous tundra vole experiment (Mustonen et al., 2002). The observed negative correlation between the plasma ghrelin levels and the cumulative food intake of the voles, however, is more enigmatic. Ghrelin is considered to induce adiposity (Tscho¨p et al., 2000), which would be observed as a positive correlation between these variables. No specific mechanism behind this phenomenon is evident in the results of this study and further experiments will be needed to explain it. The observed positive correlation between leptin and estradiol suggests that leptin could be involved also in tundra vole reproduction. This is supported by human findings with a positive correlation between plasma leptin and estradiol concentrations during and after pregnancy (Hardie et al., 1997). For ecotoxicological risk assessment it is crucial to investigate the effects of PS also in wild mammals with more susceptibility to the effects of endocrine disruptors than laboratory rodents. Our results implicate that the effects of PS are widespread. PS do not only affect circulating cholesterol concentrations or attach to steroid receptors. Instead, there are complex interactions between physiological systems requiring further studies. In previous experiments no effects of phytostanols or PS have been observed in laboratory rat reproduction. The increased probability of reproduction and decreased testesterone concentrations of male voles observed in this preliminary study could have potential for monitoring the possible effects of PS in natural ecosystems by comparing exposed populations to populations inhabiting pristine environments without e.g. pulp mill effluent discharge.
Acknowledgements We sincerely thank Mrs. Anita Kervinen and Mr. Ari Ryo¨kkynen for their invaluable help in the analyses. Mr. Ulf Hotanen provided us with the PS mixture. This work was financially supported by the Academy of Finland and by the Maj and Tor Nessling Foundation.
References Baker, V.A., Hepburn, P.A., Kennedy, S.J., Jones, P.A., Lea, L.J., Sumpter, J.P., Ashby, J., 1999. Safety evaluation of phytosterol esters. Part 1. Assessment and oestrogenicity using a combination of in vivo and in vitro assays. Food and Chemical Toxicology 37, 13– 22.
P. Nieminen et al. / Food and Chemical Toxicology 42 (2004) 945–951 Dieterich, R.A., Preston, D.J., 1977. The tundra vole (Microtus oeconomus) as a laboratory animal. Laboratory Animal Science 27, 500–506. Drexel, H., Breier, C., Lisch, H.-J., Sailer, S., 1981. Lowering plasma cholesterol with beta-sitosterol and diet. Lancet 23, 1157. Hallikainen, M.A., Sarkkinen, E.S., Gylling, H., Erkkila¨, A.T., Uusitupa, M.I.J., 2000. Comparison of the effects of plant sterol ester and plant stanol ester-enriched margarines in lowering serum cholesterol concentrations in hypercholesterolaemic subjects on a low-fat diet. European Journal of Clinical Nutrition 54, 715–725. Hardie, L., Trayhurn, P., Abramovich, D., Fowler, P., 1997. Circulating leptin in women: a longitudinal study in the menstrual cycle and during pregnancy. Clinical Endocrinology 47, 101–106. Heinemann, T., Axtmann, G., von Bergmann, K., 1993. Comparison of intestinal absorption of cholesterol with different plant sterols in man. European Journal of Clinical Investigation 23, 827–831. Hendriks, H.F., Brink, E.J., Meijer, G.W., Princen, H.M., Ntanios, F.Y., 2003. Safety of long-term consumption of plant sterol estersenriched spread. European Journal of Clinical Nutrition 57, 681–692. Hers, H.G., van Hoof, F., 1966. Enzymes of glycogen degradation in biopsy material. In: Colowick, S., Kaplan, N.O. (Eds.), Methods in Enzymology. Vol. VIII. Academic Press, New York, pp. 525–532. Jones, P.J., Raeini-Sarjaz, M., Ntanios, F.Y., Vanstone, C.A., Feng, J.Y., Parsons, W.E., 2000. Modulation of plasma lipid levels and cholesterol kinetics by phytosterol versus phytostanol esters. Journal of Lipid Research 41, 697–705. Khorasani, A.M., Cheung, A.P., Lee, C.Y.G., 2000. Cholesterol inhibitory effects on human sperm-induced acrosome reaction. Journal of Andrology 21, 586–594. Kim, J.-C., Kang, B.-H., Shin, C.-C., Kim, Y.-B., Lee, H.-S., Kim, C.Y., Han, J., Kim, K.-S., Chung, D.-W., Chung, M.-K., 2003. Subchronic toxicity of plant sterol esters administered by gavage to Sprague-Dawley rats. Food and Chemical Toxicology 40, 1569–1580. Kovacs, T.G., Voss, R.H., Megraw, S.R., Martel, P.H., 1997. Perspectives on Canadian field studies examining the potential of pulp and paper mill effluent to affect fish reproduction. Journal of Toxicology and Environmental Health 51, 305–352. Lo, S., Russell, J.C., Taylor, A.W., 1970. Determination of glycogen in small tissue samples. Journal of Applied Physiology 28, 234–236. Mattson, F.H., Grundy, S.M., Crouse, J., 1982. Optimizing the effect of plant sterols on cholesterol absorption in man. Americal Journal of Clinical Nutrition 35, 697–700. Mellanen, P., Peta¨nen, T., Lehtima¨ki, J., Ma¨kela¨, S., Bylynd, G., Holmbom, B., Mannila, E., Oikari, A., Santti, R., 1996. Woodderived estrogens: studies in vitro with breast cancer cell lines and in vivo in trout. Toxicology and Applied Pharmacology 136, 381–388. Miettinen, T.A., Puska, P., Gylling, H., Vanhanen, H., Vartiainen, E., 1995. Serum cholesterol lowering by sitostanol ester margarine in a mildly hypercholesterolemic random population. New England Journal of Medicine 333, 1308–1312. Moghadasian, M.H., 2000. Pharmacological properties of plant sterols. In vivo and in vitro observations. Life Sciences 67, 605–615.
951
Morrison, P., Dieterich, R., Preston, D., 1976. Breeding and reproduction of fifteen wild rodents maintained as laboratory colonies. Laboratory Animal Science 26, 237–243. Mustonen, A.M., Nieminen, P., Hyva¨rinen, H., 2002. Melatonin and the wintering strategy of the tundra vole, Microtus oeconomus. Zoological Science 19, 683–687. Nieminen, P., Mustonen, A.-M., Lindstro¨m-Seppa¨, P., Asikainen, J., Mussalo-Rauhamaa, H., Kukkonen, J.V.K., 2002a. Phytosterols act as endocrine and metabolic disruptors in the European polecat (Mustela putorius). Toxicology and Applied Pharmacology 178, 22–28. Nieminen, P., Lindstro¨m-Seppa¨, P., Mustonen, A.-M., MussaloRauhamaa, H., Kukkonen, J.V.K., 2002b. Bisphenol A affects endocrine physiology and biotransformation enzyme activities of the field vole (Microtus agrestis). General and Comparative Endocrinology 126, 183–189. Nieminen, P., Mustonen, A.-M., Lindstro¨m-Seppa¨, P., Ka¨rkka¨inen, V., Mussalo-Rauhamaa, H., Kukkonen, J.V.K., 2003. Phytosterols affect endocrinology and metabolism of the field vole (Microtus agrestis). Experimental Biology and Medicine 228, 188–193. Seligman, A.M., Nachlas, M.M., 1962. Lipase. In: Bermayer, H.U. (Ed.), Methoden der Enzymatischen Analyse. Verlag Chemie GmbH, Weinheim, pp. 776–778. Shintani, M., Ogawa, Y., Ebihara, K., Aizawa-Abe, M., Miyanaga, F., Takaya, K., Hayashi, T., Inoue, G., Hosoda, K., Kojima, M., Kangawa, K., Nakao, K., 2001. Ghrelin, an endogenous growth hormone secretagogue, is a novel orexigenic peptide that antagonizes leptin action through the activation of hypothalamic neuropeptide Y/Y1 receptor pathway. Diabetes 50, 227–232. Tast, J., 1966. The root vole, Microtus oeconomus (Pallas), as an inhabitant of seasonally flooded land. Annales Zoologici Fennici 3, 127–171. Tscho¨p, M., Smiley, D.L., Heiman, M.L., 2000. Ghrelin induces adiposity in rodents. Nature 407, 908–913. Van der Kraak, G.J., Munkittrick, K.R., McMaster, M.E., Portt, C.B., Chang, J.P., 1992. Exposure to bleached kraft pulp mill effluent disrupts the pituitary-gonadal axis of white sucker at multiple sites. Toxicology and Applied Pharmacology 115, 224–233. Vanhanen, H.T., Blomquist, S., Enholm, C., Hyvo¨nen, M., Jauhiainen, M., Torstila, I., Miettinen, T.A., 1993. Serum cholesterol, cholesterol precursors, and plant sterols in hypercholesterolemic subjects with different apoE phenotypes during dietary sitostanol ester treatment. Journal of Lipid Research 34, 1535–1544. Waalkens-Berendsen, D.H., Wolterbeek, A.P.M., Wijnands, M.V.W., Richold, M., Hepburn, P.A., 1999. Safety evaluation of phytosterol esters. Part 3. Two-generation reproduction study in rats with phytosterol esters—a novel functional food. Food and Chemical Toxicology 37, 683–696. Whittaker, M.H., Frankos, V.H., Wolterbeek, A.P.M., WaalkensBerendsen, D.H., 1999. Two-generation reproductive toxicity study of plant stanol esters in rats. Regulatory Toxicology and Pharmacology 29, 196–204.