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RESEARCH |
Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4231, USA
Correspondence should be addressed to M K Skinner; Email: skinner{at}wsu.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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The transgenerational disease states in the vinclozolin F1–F4 generation animals were found to be associated with a transgenerational alteration in the epigenetic (i.e., DNA methylation) programming of the male germ line (Anway et al. 2005, Chang et al. 2006). It is hypothesized that reprogramming the male germ line epigenome in subsequent generations is a mechanism by which exposure to the endocrine disruptor vinclozolin can cause a transgenerational phenotype. Epigenetic mechanisms involving DNA methylation have been shown to influence several disease states (reviews: Bjornsson et al. 2004, Egger et al. 2004). An example is that abnormal methylation of genes regulating cytokines can influence the inflammatory component of developing atherosclerosis (Corwin 2004). In addition, a change in the methylation state of the insulin-like growth factor-2 gene has been shown to increase the rate of tumor formation (Sakatani et al. 2005).
The current study was designed to investigate the actions of the endocrine disruptor vinclozolin on pregnant F0 rats and the development of transgenerational adult onset disease in their female progeny. Vinclozolin is a fungicide used in agricultural fruit crops, such as grapes for the wine industry (Kelce et al. 1994, Fisher 2004). Vinclozolin is an anti-androgenic compound that is metabolized into further anti-androgenic compounds (Kelce et al. 1994). Embryonic exposure to vinclozolin has been documented to influence sexual differentiation, gonadal formation, and reproductive functions in the exposed F1 generation as they develop and mature (Kelce et al. 1994, Gray et al. 1999, Wolf et al. 2000, Hotchkiss et al. 2002, Uzumcu et al. 2004). As discussed above, vinclozolin exposure during gonadal sex determination promotes a transgenerational epigenetic adult onset disease phenotype in males, which is transmitted through the male germ line for four generations (Anway et al. 2005, 2006a).
Observations were made that some F2 and F3 generation female offspring from the vinclozolin-treated F0 founders developed adult onset disease and had problems with their pregnancies. In late pregnancy, some rats would become pale, lethargic, and occasionally die. The current study was designed to characterize this abnormality in late-stage pregnancy and to document transgenerational adult disease states of females, which occur in the F1–F3 generation offspring of F0 pregnant rats treated with vinclozolin. The ability of an environmental toxicant to promote a variety of different disease states at high frequency for multiple generations suggests a novel epigenetic transgenerational mechanism for disease etiology.
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Results |
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9% of animals (n=105 examined).
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To better characterize the problems that were occurring with these pregnant rats, blood samples were obtained between day 19 and the day of parturition from a subset of F2 and F3 generation animals and were subjected to a complete blood count, serum chemistry analysis, or manual PCV measurements as described in the ‘In vivo procedures and treatment groups’ section of the Materials and Methods. Examination of red blood cell PCV revealed that 5 out of 35 vinclozolin generation animals were anemic (Fig. 1). PCV measures the proportion of the blood that is composed of red blood cells. These five animals were derived from three out of the five vinclozolin-treated F0 founder lines examined. None of the affected females were from the same litter. One anemic female was from the F3 generation. Nucleated red blood cells, anisocytosis (variation in red blood cell size), and polychromasia (variation in red blood cell staining) are indicative of a regenerative response to anemia. Out of the 13 vinclozolin generation animals that were subjected to a complete blood count, 2 had nucleated red blood cells present in their complete blood count, while 3 out of the 13 showed anisocytosis and polychromasia in their red blood cell morphology (derived from two out of the three founder lines examined). None of 11 control generation rats showed these abnormalities (representing three control F0 founder lines).
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The incidence of tumor formation was recorded in vinclozolin and control F1–F3 generation adult female rats. In the experimental population, 11 out of the 170 vinclozolin generation females developed tumors, compared with 3 out of 151 control generation females. This is not a statistically significant difference (P=0.10) by Fisher's exact test. The majority of the tumors identified were mammary tumors (Fig. 3C), encompassing mammary fibroadenomas, mammary adenomas, and mammary adenocarcinomas. Non-mammary tumors occurred only in the vinclozolin-treated lines, including one cerebral astrocytoma and two pituitary adenomas (Fig. 3D).
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Discussion |
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Blood analyses of F2 and F3 generation pregnant females revealed that the mean percentage of lymphocytes was decreased and that of neutrophils increased in vinclozolin generation females compared with controls. This is consistent with a ‘stress leukogram’. A stress leukogram is a decrease in the number of circulating blood lymphocytes and a corresponding increase in the percentage of segmented neutrophils, which occur when an animal experiences chronic or repeated physiological stress (Stockham & Scott 2002). Since the same set of blood cell samples for 15 different parameters were used for the complete blood analysis, a multiple testing correction suggests that the significance levels (P values) listed above must be viewed with caution. However, the fact that low lymphocyte and high neutrophil levels are consistent with the physiological condition known as stress leukogram, the finding appears physiologically relevant. The anemia phenotype seen to develop in pregnant vinclozolin generation females could easily be enough of a stressor to induce the changes in white blood cell proportions observed. Alternatively, the underlying condition that leads to the anemia phenotype might induce a stress leukogram. Therefore, the blood analysis and stress leukogram correlate with the pregnancy abnormality observed.
The blood serum chemistry profiles of F2 and F3 generation pregnant females revealed that the mean BUN, sodium, and potassium levels were significantly different (P=0.009, 0.009, and 0.039 respectively) in vinclozolin generation females compared with controls. Similar to the complete blood cell count analysis, the same set of serum samples were evaluated for 14 different serum chemistry parameters. A multiple testing correction suggests that the P values above, especially those for serum potassium (P=0.039), be viewed with caution. However, all these blood parameters are influenced by kidney function, which appear physiologically relevant. An increase in BUN, as was seen in vinclozolin generation females, suggests there are changes in the glomerular filtration that occurs in the kidneys. Therefore, the kidneys of F2 and F3 generation rats were examined histologically for signs of pathological changes. In addition to the analysis of pregnant females, the kidneys of non-pregnant females were also examined. In 70% of the vinclozolin generation females, the kidneys had a thickening of Bowman's capsules surrounding glomeruli (Ronco & Debiec 2006) in comparison with <20% in control generation females. These glomerular changes as scored for this study (see the Materials and Methods section) were mild and likely would not have resulted in complete kidney failure. Although such changes are often seen in aging rats, the age distribution of the females from whom kidneys were examined was similar in the vinclozolin and control population animals (2–10 months; data not shown). The higher proportion of abnormal kidney pathologies seen in vinclozolin generation females suggests a more rapid progression of this adult onset disease. Therefore, the kidney pathology observed correlated with the changes in BUN, sodium, and potassium seen in vinclozolin generation females. This adult onset kidney disease was seen in both non-pregnant and pregnant females.
The majority of the blood and serum chemistry parameters tested were not significantly different between control and vinclozolin generation animals (Supplementary Tables 1 and 2). For many analyses, the data for F2 and F3 generation animals were combined. A limitation of the current study is that fewer F3 generation animals were used than F2 generation animals. Major generational effects were not observed as described previously (Anway et al. 2006b), and similar observations were made between the F2 and F3 generations. Therefore, the combined F2 and F3 generation data were used to assess transgenerational phenotypes. Statistical analysis of the F1 generation animals can consider either the individual animal or litter as the experimental unit, but transgenerational F2 and F3 animals require individual animals to be the experimental unit. This accounts for biological variation between animals and phenotypes that are independent of the litter exposure.
Previous studies have demonstrated that after exposure of a pregnant F0 rat to vinclozolin during gonadal sex determination, an adult onset spermatogenic cell defect (i.e., apoptosis) developed transgenerationally in F1–F4 generation males (Anway et al. 2005, 2006a, 2006b, Anway & Skinner 2006, Chang et al. 2006). This transgenerational adult onset disease occurred in the next generation even when vinclozolin generation males were mated with control generation females. By contrast, this transgenerational spermatogenic defect phenotype was not transmitted to the next generation if vinclozolin generation females were mated with control males (Anway et al. 2005, 2006b). In the current study, it was found in 30 VOC and 15 RVOC matings that both the female and male rats had to be from vinclozolin-exposed lines in order for the anemia phenotype to occur in pregnant females of the subsequent generation. While a larger study population would be more definitive, this suggests that the proposed transgenerational epigenetic phenotype of adult onset disease induction may be different for the development of anemia during pregnancy than for those changes that influence the induction of other disease states. Results imply that the ‘inheritance’ of disease-inducing epigenetic changes is complex. Previous studies have also suggested a complex epigenetic inheritance of disease. In a study of hereditary non-polyposis colorectal cancer, it was found that abnormal hypermethylation of one allele of the DNA mismatch repair protein (MLH1) gene was associated with predisposition to the cancer. Inheritance of the affected maternal chromosome in some cases resulted in offspring also showing abnormal MLH1 methylation, while in other cases MLH1 methylation had reverted to normal (Hitchins et al. 2007). The factors that determine the inheritance of epigenetic changes are not yet known.
The signs of blood loss late in pregnancy and the glomerular pathology of the kidneys that were seen in pregnant rats in the current study are also clinical parameters of the human pregnancy disease condition pre-eclampsia (Roberts & Lain 2002, Redman & Sargent 2005, Sibai 2003, Signore et al. 2006). Another prominent parameter of human pre-eclampsia is hypertension late in pregnancy. Blood pressure was measured in the pregnant rats from F2 and F3 generation vinclozolin and control females in order to see whether the pregnancy phenotype observed in the vinclozolin generation rats closely resembled pre-eclampsia in humans. Blood pressure values were not significantly different in vinclozolin generation females, suggesting that this complex of kidney pathology and anemia seen in transgenerational vinclozolin pregnant rats may not be the same as human pre-eclampsia. Alternatively, physiological defects associated with the development of human pre-eclampsia may occur in the rats, but rats may not express hypertension as a response. Other markers of pre-eclampsia, such as platelet count and liver enzyme levels (alanine aminotransferase and alkaline phosphatase), were also not different between vinclozolin and control generation animals (Supplementary Tables 1 and 2). Further studies are necessary to elucidate the pathophysiological cause of this disease condition that occurs transgenerationally in the offspring of vinclozolin generation females.
Previous studies of the transgenerational epigenetic actions of vinclozolin on adult onset disease in males showed an increase in the rate of tumor formation in aging vinclozolin F1–F4 generations (Anway et al. 2006a). In the current study, the incidence of tumors was found to be 6.5% in vinclozolin generation animals when compared with 2% in control generation animals. This incidence of tumor formation in vinclozolin-line females is similar to what was seen in vinclozolin generation males (Anway et al. 2006a). However, in vinclozolin-line females, this 6.5% tumor incidence was not statistically different than the 2% tumor incidence seen in control-line females (P=0.10 by Fisher's exact test). Therefore, one cannot conclude that vinclozolin-line females are more prone to tumor formation than controls. In previous studies of vinclozolin-line male rats versus controls, the control-line study population had essentially 0% tumor incidence (Anway et al. 2006a). Aging female Sprague–Dawley rats are prone to spontaneous mammary tumor formation with mammary tumor incidence being as high as 60% in very old (105 weeks) females (Gitkins & Clifford 2004, Charles River Laboratories report, www.criver.com/flex_content_area/documents/rm_rm_r_lesions_survival_crlcd_sd_rats.pdf). All of the tumors seen in the current study in control-line animals were mammary tumors. Therefore, it is possible that any effect of vinclozolin treatment to increase tumor incidence was lost in the ‘noise’ of spontaneous mammary tumor formation in female rats. Further investigations with a larger study population may be needed to definitively determine whether vinclozolin treatment has an effect on mammary tumor incidence in females. Interestingly, other tumors (e.g. pituitary tumors) did develop in vinclozolin generation females but not in control generation females. These observations suggest the vinclozolin generation females, as observed in the males (Anway & Skinner 2006), may have an increased incidence of adult onset tumor formation.
The transgenerational disease phenotype in males is associated with transgenerational alterations in the epigenetic (i.e., DNA methylation) patterning of the male germ line (Anway et al. 2005, Anway & Skinner 2006, Chang et al. 2006). Although epigenetic mechanisms were not evaluated in the current study, similar transgenerational epigenetic changes are anticipated to lead to the disease phenotypes seen in vinclozolin generation female rats. When the pregnant females are exposed to vinclozolin, both the F1 generation embryos and the germ line cells that will form the F2 generation zygotes are also directly exposed to vinclozolin. Since F3 generation females show the disease phenotypes of anemia during pregnancy, white blood cell abnormalities, serum chemistry changes, and kidney abnormalities, observations indicate that the vinclozolin exposure can induce transgenerational disease states. Although the testis phenotype is transmitted through the male germ line, both male and female parents had to be of vinclozolin-exposed lines in order for female offspring to show the pregnancy disease phenotype. The extent to which the female germ line is involved in the epigenetic transgenerational effects of vinclozolin exposure remains to be elucidated.
In summary, exposure of pregnant rats to the endocrine disruptor vinclozolin resulted in the development of adult onset disease states in their female progeny. The adult onset transgenerational disease states characterized included severe anemia; a decreased percentage of lymphocytes along with an increased percentage of neutrophils; changes in serum BUN, sodium, and potassium levels; and an increased rate of renal glomerular pathology. This phenotype developed transgenerationally in females of the F1–F3 generations. These results support the findings of a transgenerational epigenetic disease phenotype in animals exposed during embryonic gonadal development to the environmental toxicant vinclozolin (Anway et al. 2005, 2006a, 2006b, Anway & Skinner 2006, Chang et al. 2006). These findings also are consistent with the hypothesis that the multi-generational effects of exposure to diethylstilbestrol, dexamethasone, or caloric restriction during pregnancy (Blatt et al. 2003, Drake et al. 2005, Zambrano et al. 2005) are in part due to epigenetic alterations. The ability of an environmental toxicant to promote a variety of different disease states for multiple generations supports the idea that a novel mechanism for disease etiology exists involving epigenetic transmission.
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Materials and Methods |
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Blood samples from 11 control rats and 13 rats from vinclozolin-treated lines (representing 3 controls and 3 vinclozolin F0 founder lines) were subjected to a complete blood count and serum chemistry analysis. PCV was measured manually as described in the Materials and Methods section for an additional 18 control-line and 21 vinclozolin-line F2 and F3 generation females to result in a total of 29 control-line and 34 vinclozolin-line animals examined (representing 5 controls and 5 vinclozolin F0 founder lines). PCV data were collected only from matched groups of control-line and vinclozolin-line females prospectively chosen for PCV analysis. For this reason, only two out of the nine females that showed the pallor and vaginal bleeding phenotype (Table 1) were included in the PCV analysis (Fig. 1), even though three out of these nine females had PCV measurements performed. All of the above-mentioned rats were of the 82 control-line and 105 vinclozolin-line rats monitored for abnormalities of pregnancy (representing 6 controls and 8 vinclozolin F0 founder lines). The animals were killed by exposing them to CO2 overdose followed by cervical dislocation in unconscious animals. The Washington State University Animal Care and Use Committee approved all procedures.
Blood cell and serum chemistry analyses
Blood analyses were performed by the Clinical Pathology Laboratory at the Washington State University Veterinary Teaching Hospital with standard procedures previously described. For the blood counts (red and white blood cells), data were collected on a Horiba ABX Hematology Analyzer System 910+CP, with all differential analysis being done manually. In some cases, packed red blood cell volume (PCV) was measured in the laboratory of the investigators using the following method: blood from lancing the lateral saphenous vein was collected into heparinized microhematocrit tubes. The tubes were centrifuged for 5 min in a Damon IEC MB microhematocrit centrifuge (Damon, Needham Hts., MA, USA), and the height of the packed red cells was expressed as a percentage of total fluid height. The metabolic panel/profile was done on a Cobras MIRA Plus Analyzer (Roche). The serum steroid concentrations were determined by RIA at the Center for Reproductive Biology Assay Core Laboratory.
Blood pressure measurements
Blood pressure was measured in awake rats placed in a restraint tube. The rats were habituated to the restraint conditions by placing each into the restraint tubes three or more times in the days prior to blood pressure measurement, until the rats remained calm in the tubes. Blood pressure was measured using an inflatable tail cuff system in combination with a Model 179 Blood Pressure Analyzer (IITC Life Sciences, Woodland Hills, CA, USA; Kubota et al. 2006). The average of two or three sequential blood pressure measurements was used.
Histology and pathology
Tissues were fixed in 10% neutral buffered formalin or Bouin's fixative (Sigma), embedded in paraffin, sectioned, and then stained with hematoxylin and eosin according to standard procedures. Multiple sections were obtained from each tissue sample for comparison to allow a representative section to be selected. The Center for Reproductive Biology, Histology Core Laboratory, and the Washington Animal Disease Diagnostic Laboratory assisted with these procedures. Renal lesions were diagnosed by an increase in morphologically identified glomerular damage exhibiting a visible fibrotic thickening of Bowman's capsule around at least half of the glomerulus, with or without accompanying reduced glomerular area. Kidneys were taken from both pregnant and non-pregnant rats 2–10 months old. Kidney sections were scored as to the extent of glomerular pathology as follows: (–)=slightly or unaffected to <10% glomeruli affected; (++)=moderately affected to 10–60% glomeruli affected; and (+++)=severely affected to >60% of glomeruli affected. Note that slightly and moderately affected individuals would likely show no clinical signs of kidney insufficiency due to excess kidney capacity.
Statistical analysis
Results from vinclozolin and control generation group animals were compared using Student's t-test or t-test with Welch's correction for non-equal variances for parametric data and Fisher's exact test for non-parametric data sets. In the case of analyses of combined F2 and F3 data for neutrophils, lymphocytes, BUN, sodium, and potassium, a two-way ANOVA indicated no significant effect of generation. Therefore, the F3 data were pooled with that of F2 for several parameters. Analyses were performed using GraphPad Prism 4 version 4.01 for Macintosh (GraphPad Software, San Diego, CA, USA).
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Acknowledgements |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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Received 5 December 2007
First decision 2 January 2008
Accepted 24 January 2008
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References |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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