The Toxicologist - Society of Toxicology

median CR-adjusted isoflavone concentrations were higher than those in the U.S
reports, but the lignans concentrations were lower than those in the U.S reports.
The CR-adjusted urinary concentrations of SEC, GEN and DAI of infertile group
were significantly higher than those of control group. With the increasing SEC,
GEN and DAI levels (assessed as quintiles), the odds ratios (ORs) of these semen
parameters showed the similar trends. From the first quintile to the fourth quintile,
there were no significant variations about the ORs, whereas from the fourth quintile
to fifth quintile, the ORs increased significantly. Subjects with higher urinary
concentrations of SEC, GEN and DAI were more likely involved in idiopathic
male infertility with abnormal semen quality (P < 0.01, 0.05 and 0.05, respectively).
Conclusion: High level urinary concentrations of SEC, GEN and DAI were
found associated with increased risk of male idiopathic infertility. Furthermore, the
idiopathic infertile subjects with abnormal semen might be in higher risk. These
findings should be of concern because of the ubiquitous exposure of PEs.
391 REPRODUCTIVE AND DEVELOPMENTAL TOXICITY
OF DEGRADATION PRODUCTS OF REFRIGERANTS.
M. Ema, M. Naya, K. Yoshida and R. Nagaosa. Research Institute of Science for
Safety and Sustainability, National Institute of Advanced Science and Technology
(AIST), Tsukuba, Ibaraki, Japan.
We summarized the findings of recent animal studies on the reproductive and developmental
toxicity of the degradation products of 1,1,1,2-tetrahydrofluoroethane
(HFC-134a) used worldwide and 2,3,3,3-tetrafluoropropene (HFO-1234yf) developed
for new generation. Trifluoroacetic acid (TFA), carbon dioxide (CO2), carbon
monoxide (CO), carbonyl fluoride (CF), hydrogen fluoride (HF) and formic
acid (FA) were evaluated . Excessive CO2 is testicular and reproductive toxic, embryolethal,
developmentally neurotoxic and teratogenic in experimental animals.
Maternal exposure to CO causes prenatal and postnatal lethality and growth retardation,
skeletal variations, cardiomegaly, blood biochemical, immunological and
postnatal behavioral changes, and neurological impairment in offspring of several
species. In very early studies, CO was reported to be teratogenic in rats and guinea
pigs. The results of toxicological studies on sodium fluoride (NaF) were used to obtain
insight into the toxicity of CF and HF, because CF is rapidly hydrolyzed in
contact with water yielding CO2 and HF, and NaF is similar in kinetics and dynamics
to HF. Increased fetal skeletal variation, but not malformation, was noted
after the maternal administration of NaF. Rat multiple-generation studies revealed
that NaF caused retarded ossification and degenerative changes in the lung and kidney
of offspring. There is a lack of information about the toxicity of TFA and FA.
Animal studies remain necessary for the risk assessment of chemicals because it is
difficult to find alternative methods to determine the toxic effect of chemicals.
392 DETERMINANTS OF PREGNANCY SUCCESS IN THE
CYNOMOLGUS MONKEY.
C. M. Luetjens and G. F. Weinbauer. Covance Laboratories GmbH, Muenster,
Germany.
Although an age-effect on reproduction outcome is well documented, information
on relevant parameters for a successful mating programme in macaque species is
scarce. Thus, we conducted a cohort study on cumulative pregnancy rates in the
cynomolgus monkey, currently the predominant nonhuman primate model for
preclinical safety assessment. A retrospective analysis was performed on 3870 matings
with 1489 females and 82 males. Ovarian cycles were monitored and animals
were cohabitated during the anticipated ovulation event. Pregnancy rates were calculated
by comparing different cohorts (n=35) of females over the last 4 years
grouped for age and body weight at mating start. Single (first time)-mated females
were compared with multi-mated females to assess bias of several mating attempts
per female. Analysis of male mating success was also included. Data were explored
according to age, season, and weight of both sexes. Overall, 376 pregnancies
(25.7%) were achieved in single-mated females. For multi-mated females, 946
pregnancies (65% per animal, 38% per ovarian cycle) were attained during 2.4 ±
1.7 (mean ± SD) matings. Subgroup analysis by age revealed a cumulative pregnancy
rate of 0% below 3.0 years, 18.2% until 3.5 years, 20.9% until 4.0 years,
25.3% until 4.5 yrs and 30.4% until 5.0 years and older. Pregnancy age distribution
was independent of number of matings and the age distribution at first mating
was comparable. Female body weight at mating was of minor relevance for pregnancy
success except for females exceeding 5.2 kg with a 50% reduced pregnancy
rate. The males used in the study had an age range of 5.2 to 14.5 years with a median
of 7.6 years and a weight range of 5.3 to 14.1 kg with a median of 7.6 kg. Male
factors contributing to the pregnancy outcome - besides full spermatogenesis and
ejaculated sperm - could not be identified. In summary, unlike age, neither female
body weight, different male partners, nor season had a significant effect on pregnancy
rates. This study demonstrates the impact of female age on colony breeding
outcome. Healthy female animals older than 3 years are mandatory for a successful
mating programme.
393 GENE ALTERATIONS UNDERLYING 2, 5-
HEXANEDIONE-MEDIATED SUPPRESSION OF
RADIATION-INDUCED GERM CELL APOPTOSIS.
S. N. Campion 1 , M. A. Sandrof 1 , E. Houseman 2 , J. Hensley 3 , Y. Sui 2 , K. W.
Gaido 3 , Z. Wu 2 and K. Boekelheide 1 . 1 Pathology and Laboratory Medicine, Brown
University, Providence, RI, 2 Community Health, Brown University, Providence, RI
and 3 The Hamner Institutes for Health Sciences, Research Triangle Park, NC.
Sertoli cells are essential for testicular germ cell maintenance and survival. We made
the unexpected observation that x-radiation (x-ray)-induced germ cell loss is attenuated
by co-exposure with the Sertoli cell toxicant 2,5-hexanedione (HD). The cellular
and molecular mechanisms underlying this attenuation of germ cell apoptosis
with reduced Sertoli cell support are unknown. The current study was performed to
examine alterations in testicular gene expression with co-exposure to HD and x-ray.
Adult male rats were exposed to HD (0.33% or 1%) in the drinking water for 18
days followed by x-ray (2Gy or 5Gy), resulting in a total of 9 treatment groups.
Testis samples were collected after 3 hr and gene array analysis was performed.
Using a novel bioinformatic approach to summarize the effect of HD across all
treatment groups, we focused on the modification of x-ray-induced gene alterations
by HD co-exposure. Gene Set Enrichment Analysis was used to identify biological
pathways where HD modification of gene expression was the greatest. HD exerted
a significant influence on genes involved in Cell Cycle and DNA Replication,
Recombination, and Repair. HD also had an antagonistic effect on x-ray-induced
alterations of several apoptotic genes (Fas, BBC3, AEN). To further investigate the
specific cell populations and stages in which these critical gene alterations occur,
laser capture microdissection (LCM) samples were collected from the basal compartment
of the seminiferous epithelium, enriching for those germ cells most susceptible
to x-ray-induced apoptosis. Quantitative RT-PCR of the LCM samples
confirmed the suppression of apoptotic genes by HD co-exposure. The results of
this study provide insight into the mechanisms underlying attenuated germ cell
toxicity following HD and x-ray co-exposure and also provide a novel bioinformatic
approach to analyzing co-exposure effects on gene expression.
394 GENE EXPRESSION CHANGES IN THE TESTIS
FOLLOWING X-RADIATION EXPOSURE.
N. Catlin, S. N. Campion, M. A. Sandrof, S. J. Hall and K. Boekelheide.
Pathology and Laboratory Medicine, Brown University, Providence, RI.
Spermatogenesis is controlled by paracrine-interacting cell types, including Sertoli
cells and germ cells within the testis. Testicular toxicants can target specific cells
within the testis, altering molecular pathways and leading to cellular damage, such
as germ cell apoptosis and spermatogenic failure. X-radiation (x-ray) directly induces
germ cell apoptosis; however, this apoptotic response is attenuated by co-exposure
to the Sertoli cell toxicant, 2,5-hexanedione (2,5-HD). By analyzing gene
expression in response to x-ray and 2,5-HD exposure we have identified molecular
changes underlying this attenuated toxicity. Previous research in our laboratory utilized
microarray analysis to determine apoptosis-related genes induced in the testis
by 2Gy and 5Gy x-ray, doses which elicit overt toxicity. These genes include BCL2
Binding Component 3 (BBC3), Apoptosis Enhancing Nuclease (AEN) and
Caspase 3. To investigate these gene responses at low doses of x-ray, rats were exposed
to 0Gy, 0.5 Gy, 1 Gy and 2Gy x-ray, sacrificed 3 hours after exposure, and
the testes were removed. Total RNA was extracted from the isolated testes and the
expression of BBC3, AEN and Caspase 3 was measured by real time PCR. A doseresponse
in gene expression was observed, with all 3 genes exhibiting the highest expression
at 2Gy, decreasing slightly as the dose was reduced to 0.5Gy. Having investigated
the apoptotic gene responses to low dose x-ray exposure alone, rats will
be co-exposed to x-ray and the Sertoli cell toxicant 2,5-HD in the low dose range
and gene alterations will be measured by microarray and real time PCR. We hypothesize
that low-dose extrapolation of 2,5-hexanedione and x-radiation co-exposure
will distinguish adaptive from adverse gene alterations and elucidate the mechanism(s)
underlying the phenotypic consequences of co-exposure.
395 EFFECT OF METHOXYCHLOR ON OVARIAN ANTRAL
FOLLICLES FROM ESTROGEN RECEPTOR ALPHA
(ESR1) OVEREXPRESSING MICE.
T. Paulose, I. Hernández-Ochoa and J. A. Flaws. Veterinary Biosciences, University
of Illinois Urbana-Champaign, Urbana, IL.
Methoxychlor (MXC) is an organochlorine pesticide that was used worldwide to
protect crops, fruits and vegetables. Although banned in the USA, MXC still persists
in the environment and the general population continues to be exposed to it at
various levels. Current studies demonstrate that MXC causes adverse ovarian toxicity
by inhibiting growth and causing atresia of antral follicles, which are the functional
units of the ovary. However, the exact mechanistic pathway by which MXC
SOT 2010 ANNUAL MEETING 85