ToxSci Advance Access originally published online on September 22, 2007
Toxicological Sciences 2007 100(2):504-512; doi:10.1093/toxsci/kfm245
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Reversibility of the Adverse Effects of 1-Bromopropane Exposure in Rats
* Department of Occupational and Environmental Health 466-8550
Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya, 466-0065 Japan
School of Public Health, Anhui Medical University, Hefei, 230032 China
Department of Molecular Neurobiology, Institute for Developmental Research, Aichi Human Service Center, Kasugai, 480-0392 Japan
¶ Shanghai Institute of Planned Parenthood Research, Shanghai, 200032 China
|| Emeritus Professor, Nagoya University School of Medicine, Nagoya, Japan
||| Center for Reproductive Medicine, Toyohashi Municipal Hospital, Toyohashi, 441-8570 Japan
|||| Department of Human Functional Genomics, Life Science Research Center, Mie University, Tsu, 514-8507 Japan
1 To whom correspondence should be addressed at Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Fax: +81-52-744-2124. E-mail: gak{at}med.nagoya-u.ac.jp.
Received June 24, 2007; accepted September 17, 2007
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Previous experiments indicated that 1-bromopropane (1-BP), an alternative to chloroflurocarbons, is neurotoxic and inhibits spermiation in the testis. Here we investigated the reversibility of the toxic effects of 1-BP in rats. Male Wistar rats were divided into three equal groups of 24 each and exposed by inhalation to 0, 400 or 1000 ppm of 1-BP for 6 weeks (8 hrs/day, 7 days/week). Eight rats from each group were sacrificed at the end of 6 weeks exposure, and at 4 and 14 weeks after the end of exposure, to assess the recovery processes. We studied sperm count, motility, morphology and testicular histopathology, as well as blood pressure, skin temperature and hindlimb muscle strength. At the end of 6 weeks of exposure to 1000 ppm (0 week recovery), testicular weight, epididymal weight, sperm count and motility were low, morphologically abnormal sperm were increased and spermatogenic cells showed diffuse degeneration. These changes did not show full recovery at 14 weeks recovery, with the exception of the prostate and seminal vesicular weights, which recovered back to control values. At 400 ppm, increased retained spermatids at 0 week recovery returned to normal at 4 weeks recovery. Exposure to 1000 ppm produced sustained reduction of hindlimb muscle strength at 14 weeks recovery, whereas normalization of the skin temperature and blood pressure was noted after transient changes. Our study showed that the effect of 1-BP on spermatogenesis is dose-dependent; low exposure inhibited spermiation and hormone-dependent organ weight reduction and these changes were transient, while a higher dose of 1000 ppm 1-BP caused persistent depletion of spermatogenic cells.
Key Words: neurotoxicity; reproductive toxicity; 1-bromopropane; spermatogenic cells; spermiation; 
-enolase.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Bromopropanes have become alternatives to chloroflurocarbons and 1,1,1,trichloroethane, which is known to deplete the ozone layer (Ichihara 2005
). 2-Bromopropane (2-BP) was the first bromopropane to be used as an alternative to ozone-depleting solvents in Korea and Japan (Takeuchi et al., 1997). However, it was subsequently found to be toxic to reproductive and hematopoietic organs in humans (Kim et al., 1996) and animals (Ichihara et al., 1997; Ichihara et al., 1996; Yu et al., 1997), and was later banned. Its isomer, 1-bromopropane (1-BP) has been used as an alternative (Ichihara 2005) in spray adhesives and as a cleaner and degreaser. It is also used as an intermediate in the synthesis of various pharmaceuticals, insecticides, flavors and fragrances and as a solvent for fats, waxes or resins (NTP Center et al., 2004).
In a series of studies, we found 1-BP to exhibit dose-dependent neurotoxicity manifested by deterioration of distal latency and motor nerve conduction velocity of tail nerve as well as weakness of muscle strength of forelimbs and hindlimbs in rats exposed to 1-BP at 200, 400, and 800 ppm for 12 weeks (Ichihara et al., 2000a). We also found a dose-dependent decrease in gamma-enolase in the cerebrum of rats exposed to 1-BP for 7 days (Wang et al., 2002) and 12 weeks (Wang et al., 2003). 1-BP also exhibited reproductive toxicity; exposure to 200, 400, and 800 ppm for 12 weeks decreased epididymal sperm count and sperm motility in a dose-dependent manner along with an increase in the number of retained elongated spermatids (step 19) in the seminiferous epithelium of rat testes at postspermiation stages (Ichihara et al., 2000b).
The present study was an extension to the above studies and designed to determine the reversibility of the aforementioned toxic effects of 1-BP upon its withdrawal and the correlation of any such changes with the exposure level. We exposed male Wistar rats to 1-BP for 6 weeks and evaluated the recovery of the reproductive and central nervous systems at 4 and 14 weeks after withdrawal. In addition, we also studied tail blood pressure and tail skin temperature, since 1-BP is supposed to cause autonomic nervous system disturbances (Ichihara 2005).
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Animals and exposure to 1-BP.
The study was conducted in 72 specific pathogen-free 8-week-old male Wistar rats purchased from Shizuoka laboratory animal center, Japan. They were housed and acclimatized to the new environment for 2 weeks, then divided equally at random into three groups (0, 400, and 1000 ppm, n = 24, each). They were housed in a room set on 16:8-h light:dark cycle (lights on at 0600 h and off at 2200 h), stable relative humidity (67–60%) and constant temperature (23°C–25°C). Food and water were provided ad libitum. Body weight was measured between 8.00 to 9.00 am once a week. The rats of each group were exposed to 1-BP for 8 h/day for each day of the week for 6 weeks. The inhalation exposure system used in the present study has been described previously (Ichihara et al., 1997
; Takeuchi et al., 1989). In brief, a regulated volume of 1-BP was evaporated at room temperature and mixed with a larger volume of clear air to achieve the target concentrations. The vapor concentration of 1-BP in the chamber was measured every 10 sec by gas chromatography and electronically controlled to within ± 5% of the target dose. The mean concentration measured every 10 sec for 8 hours was considered the value for that day. This was then averaged over 6 weeks in order to obtain the mean and standard deviation, which was 995 ± 40 and 997 ± 34 in the 1000 ppm chambers and 410 ± 19 and 405 ± 24 in the 400 ppm chambers. Rats of all three subgroups, namely 0, 4, and 14 weeks recovery were equally represented in the respective exposure chambers .1-BP (99.8% purity) was kindly supplied by Tosoh Co., Japan. The Japanese law concerning protection and control of animals and the Guide of Animal Experimentation of Nagoya University School of Medicine were followed throughout the experiment.
At the end of the 6-week exposure period, eight rats from each group were sacrificed to examine the effects of exposure to 1-BP. The remaining 16 rats in each group were allowed to recover. At the end of 4 weeks after withdrawal, eight rats from each group were sacrificed to examine the recovery changes. The remaining eight rats from each group were sacrificed and examined at 14 weeks after termination of exposure.
Measurement of hindlimb muscle strength (HLMS).
HLMS was measured once a week at 9.00–10.00 from the end of 6 weeks of exposure until 5 weeks after cessation of exposure. It was then measured every 2 weeks until the end of the study. HLMS was measured using a push–pull scale (Imada Co., Japan) according to the method described by Meyer et al (Meyer et al., 1979). Animals were made to climb a ladder-like metallic transverse bars attached to the pushing side of the scale. By pushing down the transverse bars, the maximum strength of hindlimb muscles was recorded. The average of three readings was considered the hindlimb muscle strength for that rat at the specified period.
We did not measure nerve conduction velocity in the tail nerve to avoid any possible injury to the nerve, which in turn might affect recovery.
Measurement of tail skin temperature.
This was measured in the awake rats in the dorsal part of the rat tail about 2 cm from the tail tip using a digital temperature recorder (model BDT-100, BRC) with a sensitive probe at the end of a plug-in-wire. The tail temperature was recorded after the temperature reading in the digital thermometer remained stable for 1–2 min.
Measurement of tail blood pressure.
Systolic blood pressure (SBP) of conscious rats was measured by the tail cuff method using BP-98A, MCP-1 (Softron, Tokyo, Japan) as described previously (Ichihara et al., 2007) . The blood pressure values reported in this study represent the mean of four or five determinations taken at the same period.
Organ weights and histopathological examination.
After exposure for 6 weeks and the following 4 or 14 weeks postexposure, eight rats from each group were sacrificed by decapitation and blood was collected in heparinized tubes. The blood was centrifuged and the plasma was stored at – 80°C until analysis. The brain was quickly dissected out and the cerebrum, cerebellum, midbrain, pons, hippocampus, caudate putamen, amygdala, medulla oblongata and the remaining parts containing thalamus and hypothalamus were cut and stored at – 80°C until analysis. The reproductive organs of the epididymis, testis, prostate and seminal vesicle were dissected out and weighed. The testis and epididymis were fixed in Bouin's solution while other organs were fixed in 10% neutral buffered formalin. The organs were embedded in paraffin and cut into 5-µm-thick sections. Tissue sections of the testis were reacted with Periodic acid-Schiff and counterstained with hematoxylin, while other organs were stained with hematoxylin and eosin and observed under light microscope. The same procedure was repeated at the end of 4 and 14 weeks after withdrawal of 1-BP.
Epididymal sperm count, motility and morphological abnormalities of sperm head.
Sperm were collected as quickly as possible after sacrifice. The right cauda epididymidis was pierced at two locations using fine forceps and slight pressure was applied to the structure to collect the sperm-containing fluid into a dish containing 2 ml of Hank's solution. The sperm suspension (5 µl) was used to study sperm motility. Progressive or nonprogressive motile sperm were counted by a computer-assisted sperm analysis (CASA) system (C-IMAGING C-MEN, Compix Inc., Cranberry township, PA, 16066, USA). The remainder of the right cauda epididymidis was minced in Hank's solution and filtered through a gauze. The sperm suspension obtained after mincing of the cauda epididymidis was used for total sperm count and morphological examination of sperm head; it was first diluted with 0.5% formalin solution and then infused into a hemocytometer to count the number of sperm under a microscope. Furthermore, a smear was prepared from this suspension on a glass slide to study the sperm head morphology under a phase contrast microscope. Sperm with morphologically abnormal heads were counted among 300 sperm in total. The head morphology was classified as straight, banana like, teratic (amorphous or pyknomorphous) and Unclassified head according to Mori et al (Mori et al., 1991).
Retained spermatids in seminiferous tubules.
Retained elongated spermatids and nuclei of Sertoli cells were counted in 12 round or ovoid seminiferous tubules at postspermiation stages (stage IX, X, and XI), which were randomly selected from one section of the testis, by direct observation under a light microscope using the method described previously (Ichihara et al., 2000b). When the total number of tubules at stage IX, X, and XI was less than 12, all tubules at stage IX–XI were used for counting. Only retained elongated spermatids near the basement membrane were counted for quantitative evaluation. The criterion was that more than half of the profile should be in the basal 1 or 2 layer (layer of leptotene or pachytene spermatocytes). Stages of the cycle in rats were classified according to Russell et al (Russell et al., 1990). Counts of retained elongated spermatids were determined only in the 400 ppm and control groups since the testes of rats exposed to 1000 ppm showed pyknotic nuclear changes in the round spermatids, making stage identification impossible or unreliable. The number of retained spermatids was expressed per tubule and per 100 Sertoli nuclei. A mean value of 12 tubules was considered as the representative value of each animal.
Hormone assay.
Plasma samples were stored at – 80°C until assayed for testosterone. Plasma testosterone levels were measured by SRL, Inc. Tachikawa, Tokyo, Japan, using a radioimmunoassay kit (DPC total testosterone kit, Simens Medical Solutions Diagnostics, Tarrytown, NY).
Immunoassays of nerve-specific marker proteins and gamma enolase.
Since gamma enolase is localized specifically in neurons, a decrease in the concentration of this enzyme reflects a decrease in the enzyme amount per cell or a decrease in the number of neurons, suggesting adverse effects on neurons. In our previous studies, low levels of neuro-specific gamma enolase were found in the cerebrum after exposure to 1-BP at 400 and 800 ppm for 12 weeks (Wang et al., 2003) and for 7 days (Wang et al., 2002). Accordingly, we measured gamma-enolase in the present study as part of our investigation of recovery from 1-BP neurotoxicity. In the present study, the brain was dissected into smaller regions compared with our previous studies (Wang et al., 2002; 2003). Tissue blocks of various brain regions were homogenized in 10 volumes (wt/vol) of 50mM phosphate buffer (pH 7.4) containing 5mM ethylenediaminetetraacetic acid at 0°C. The homogenate was centrifuged at 45,000 x g for 20 min at 4°C. The supernatant was used for gamma enolase analysis. Gamma enolase was determined by a highly sensitive sandwich type enzyme immunoassay system developed by Kato et al. (1981). The protein concentration of the soluble fraction of homogenate was estimated by the dye-binding method of Bradford (Bradford 1976) using Bio-Rad (Hercules, CA) reagents.
Statistical analysis.
Data were expressed as mean ± SD. Differences between the exposure and control groups were examined for statistical significance using Dunnett's method following one-way ANOVA. The percentage values were converted by arcsine transformation before the above analysis. Log transformation was performed before ANOVA for the number of retained spermatids to equalize the variance, because the standard variation increased in proportion to the mean value. A p value less than 0.05 denoted the presence of a statistically significant difference.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Body Weight
The body weight of rats exposed to 1000 ppm was significantly lower than the control from 1 week after the beginning of exposure until 7 weeks after the end of the exposure (Table 1). The body weight of rats exposed to 400 ppm was not significantly different from the control throughout the experiment.
|
Weight of Reproductive Organs
One control rat died during blood pressure measurement at week 5 of exposure and one rat of the 1000 ppm group died 2 days after completion of the 6 weeks exposure due to severe debilitation.
The weight of the testis was significantly lower in the 1000 ppm group than the control at 0 week recovery, and did not show any recovery by postexposure week 4 and 14 (Fig. 1). The weight was 70% of the control value at postexposure week 0 following 6 weeks exposure to 1000 ppm, and further decreased to 36% of the control values at the end of the recovery period of 14 weeks. Similarly, the weight of the epididymis in rats exposed for 6 weeks to 1000 ppm was significantly lower than the control at 0 week recovery and remained lower without any recovery at the end of the 14-week recovery period (Fig. 2). On the other hand, the weight of the prostate of rats exposed for 6 weeks to 1000 ppm was significantly lower at postexposure week 0, but improved gradually and reached the control value at the end of the 14-week recovery period (Table 1). Furthermore, the weight of the seminal vesicle of rats exposed for 6 weeks to 1000 ppm was also significantly lower at postexposure week 0 but recovered dramatically 4 weeks after withdrawal of 1-BP (Table 1).
|
|
Testosterone Levels
Plasma testosterone was significantly lower in the 1000 ppm group than the control at 0 week recovery (Table 1) but recovered to the control level at 4 weeks recovery.
Sperm Count and Motility
The epididymal sperm count was significantly reduced in both the 400 and 1000 ppm groups at 0 week recovery following 6 weeks of exposure (Table 2). However, after the 4-week recovery period, the sperm count increased to normal level in the 400 ppm exposed rats but tended to decrease further in the 1000 ppm rats, and remained significantly reduced even at 14 weeks after the end of exposure. Thus, the sperm count, which was 8% of the control value following exposure, progressively decreased to 1.5% of the control during the recovery period. In the 1000 ppm group, the sperm motility of 62.6% (which is 77% of the control value) at 0 week recovery decreased further to 45.9% (59% of the control) at the end of the recovery period of 14 weeks (Table 2).
|
Sperm Morphology
Following 6 weeks exposure to 1-BP at 1000 ppm, the percentages of sperm with normal head was lower and those with abnormal heads, e.g., teratic head (amorphous and pyknomorphous heads), straight head and banana head, were significantly higher than 0 ppm (control) (Table 2). At 4-week recovery, the percentages of sperm with teratic head, straight head, banana head and unclassified head (abnormal heads that could not be classified) were further increased along with further decrease in sperm with normal head in the 1000 ppm group. At 14 weeks recovery, the percentages of sperm with straight head, teratic head, banana head and unclassified head in the 1000 ppm group remained significantly high, together with low percentage of sperm with normal head.
Histopathological Findings
Testis.
Control rats showed normal histological structure of the testis at 0, 4, and 14 (Fig. 3A) weeks recovery. In rats exposed for 6 weeks to 1-BP at 1000 ppm, the seminiferous tubules showed the following changes at 0-week recovery, (1) formation of multinucleated giant cells, (2) Sertoli cell vacuolation, (3) diffuse spermatogenic cell degeneration, and (4) round spermatids with pyknotic nuclei (Fig. 3B). On the other hand, in rats exposed for 6 weeks to 400 ppm, changes were mild except for an increase in the number of retained elongated spermatids at post spermiation stages IX–XI (Fig. 3E). The number of retained elongated spermatids at the basal region of the post spermiation stage tubules of the 6-week 400 ppm 1-BP–exposed rats was significantly higher at 0 week recovery compared with the control (0 ppm), but normalized at 4 weeks after cessation of exposure (Table 3).
|
|
Further examination of the rats exposed to 1000 ppm and allowed to recover for 4 weeks showed lack of recovery and deterioration with degeneration of almost all spermatogenic cells along with atrophy of the seminiferous tubules (Fig. 3C). The majority of the seminiferous tubules showed depletion of spermatogenic cells. The tubules were markedly shrunken and hardly contained any differentiated spermatogenic cells (Fig. 3C). After recovery for 14 weeks, the seminiferous tubules showed little recovery in the testes of the 1000-ppm–exposed rats; which typically showed very few spermatogenic cells (Fig. 3D). The tubular lumen was deficient in sperm (Fig. 3D). The tubules showed further shrinking compared to the 4 weeks recovery group.
Epididymis.
In the epididymis of 1000 ppm rats at 0 week recovery, the duct diameter was smaller, the number of sperm in the duct cavity was lower, and the epithelial layer was thicker than the control. Furthermore, neutrophils and degenerated epithelial cells were frequently found in the duct cavity. At 4 week recovery in the 1000 ppm group, empty space occupied a larger part of the epididymal duct cavity, which contained very few sperm and was lined with smaller diameter and taller epithelium, than the 0 week recovery epididymis. At 14 weeks recovery, almost the entire duct cavity was empty and the diameter of the duct and epithelial height showed no recovery, and only a few of the duct cavities contained neutrophils. In the 400 ppm group, the epididymal duct cavity contained more neutrophils and degenerated cells than the control at 0 and 4 weeks recovery. However, the histological findings were similar to the control at 14 weeks recovery.
Seminal vesicle.
In the 1000 ppm group at 0 week recovery, the epithelial layer was reduced in thickness compared with the control, secretory granules or vesicles inside the epithelial cells were reduced in number, and degenerated cells (probably derived from the epithelium) were found in the vesicular cavity. At 4 weeks recovery, much of the vesicular epithelium and cavity showed recovery, and at 14 weeks, recovery of the seminal vesicle epithelium and vesicular cavity was almost complete. On the other hand, there were no differences between the control and 400 ppm group at 0, 4, and 14 weeks recovery.
Prostate.
In the 1000 ppm group at 0 week recovery, the thickness of the prostate epithelium was less and the cavity was smaller than the control. However, both variables returned to the control levels at 4 and 14 weeks recovery. The prostate of the 400 ppm group at 0 week recovery did not show any remarkable changes in epithelial cells but the cavity was smaller than the control, although it returned to the control at 4 and 14 weeks recovery.
Rat tail skin temperature.
The rat tail skin temperature was significantly low in the 1000 ppm group at recovery week 0 following 6 weeks of exposure to 1-BP but the temperature returned to control levels at 4 weeks of recovery (Table 4).
|
Rat tail blood pressure.
The tail blood pressure in rats exposed for 6 weeks to 1000 ppm remained significantly elevated starting from 4 weeks of exposure to 5 weeks of recovery. At 8 weeks after recovery, the blood pressure returned to the same values compared to the control group (Table 5).
|
Hindlimb muscle strength.
Rats exposed to 1000 ppm tended to sit with their legs stretched most of the time and were unable to stand up steadily on their hind limbs to feed. When allowed to walk, they refused to move and when stimulated to move, they tended to drag their hindlimbs rather than walk step-by-step. The hindlimb muscle strength in these rats was significantly diminished at 0 week recovery and remained low with no recovery even at 14 weeks after stopping exposure to 1-BP (Fig. 4).
|
Gamma enolase in the brain.
In the present study, no significant changes were noted in gamma enolase determined in the different regions of the brain in all exposure groups (data not shown).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
The adverse effects of 1-BP on the reproductive system should be described as a two-phase pattern. A low level exposure at 400 ppm caused failure of spermiation, while a high level exposure at 1000 ppm induced a completely different and severe effect in the form of spermatogenic cell depletion, rather than just a dose-dependent increase in the number of retained spermatids. Many toxic agents are known to produce retained spermatids out of which biphasic changes were also observed in boric acid (Ku et al., 1993
).
Exposure to 1-BP at 400 ppm resulted in reduction of epididymal sperm count after the end of exposure, accompanied by an increase in sperm retention at post spermiation stages. We also observed in our previous study of 12 weeks exposure to 1-BP at 200, 400, and 800 ppm (Ichihara et al., 2000b) a dose-dependent increase in the number of retained spermatids, together with a dose-dependent decrease in the epididymal sperm count. Based on these findings, we indicated that the increase in retained spermatid might explain the decrease in epididymal sperm count. In the present study, such interpretation is more strongly supported by the fact that the recovery of the epididymal sperm count at 4 weeks in the 400 ppm group was also accompanied by normalization of sperm retention. Omura et al. (1996) also showed retained and degenerated sperm in the basal region of seminiferous tubules at postspermiation stages in the testis of the rats administered gallium arsenide. They speculated that some malformed spermatids due to disturbance of spermiation could not be released from the seminiferous epithelium and were phagocytosed by Sertoli cells, thereby increasing the number of retained spermatids.
On the other hand, histopathological studies showed severe atrophic changes in the seminiferous tubules following exposure to 1000 ppm of 1-BP and such changes were progressive. Our previous study (Ichihara et al., 2000b) showed no changes in the number of spermatogonia, spermatocytes and round spermatids after exposure to 1-BP at 800 ppm, eight hours per day, seven days per week for 12 weeks. In that study, we discussed the major differences in the targets of 1-BP and 2-BP, namely that 1-BP inhibits spermiation while 2-BP targets spermatogenic cells. However, the present results necessitate modification of that statement. We justify this statement under exposure to levels 800 ppm or below, but it should be pointed out also that overexposure to 1-BP results in damage and depletion of spermatogenic cells. The critical level at which spermatogenic cell depletion is induced by 1-BP should be explored further. The progressive testicular atrophy noted in our study was associated with deteriorating histopathological changes in the epididymis and a progressive decrease in testicular weight, epididymal weight, sperm count and sperm motility and lasted longer than other temporary changes such as spermiation failure, decrease in plasma testosterone level and abnormal histopathological changes and weight reduction of hormone-dependent seminal vesicle and prostate.
The recovery of 2-BP–induced testicular atrophy is also reported to be dependent on the dose level, duration of exposure and recovery period (Lee et al., 1998; Leone et al., 1988; Saegusa 1989; Shemi et al., 1982; Son et al., 1997; Son et al., 1998, 1999). In the present study using 1-BP, rats exposed to a lower dose (400 ppm) showed a better recovery than those exposed to 1000 ppm, which showed no recovery, indicating a dose-dependent response, but it is possible that a shorter duration of exposure at 1000 ppm could produce reversible changes. Further studies using different exposure periods and recovery periods are required to investigate this possibility.
With regard to the effects of exposure to 1000 ppm of 1-BP on the central nervous system and subsequent recovery, rats exposed to this high dose showed a number of physical signs of stress such as reduction in body weight, debilitation, irritability, weakness and severe hind limb muscle weakness. These results are in agreement with those of previous studies, which showed reduction of hindlimb grip strength (Ichihara et al., 2000a). Based on these results, we expanded the present study to assess the recovery of hindlimb muscle strength following exposure to 1-BP for 6 weeks. Such exposure resulted in a significant reduction of hindlimb muscle strength and the strength did not improve to control level even at the end of the 14-week recovery phase. In this regard, humans exposed to 1-BP also reported lower limb weakness, variable degrees of difficulty on standing or walking and numbness in the lower extremities bilaterally (Harney et al., 2003; Ichihara et al., 2002). Furthermore, we reported previously in an experimental study that rats exposed to 1-BP had low levels of glutathione (GSH) in the brain (Wang et al., 2002; Wang et al., 2003) and since such deficit can cause oxidative stress, which is linked with hindlimb weakness (Jamie et al., 2006), GSH depletion might explain the neurotoxicity of 1-BP. However, further studies are needed to confirm such mechanism of action. Further studies are also needed to clarify whether the persistent deficit in hindlimb muscle strength is related to changes in the central nervous system, peripheral nerves or muscles.
The thermal control of rat tail blood flow, which resembles that of the human forearm and finger (Wenger et al., 1975), recovered to baseline level at recovery week 4, indicating that the recovery of peripheral vasoconstriction mechanisms is the fastest of all other nervous system-related disturbances, namely the elevated blood pressure and persistently low hindlimb muscle strength.
We reported previously that exposure to 1-BP at 800 ppm, eight hours per day, seven days a week, for 12 weeks decreased the cerebral levels of gamma enolase, a neuron-specific protein (Wang et al., 2003). The cerebral region examined in that study included amygdala, hippocampus, cortex and caudate putamen collectively. However, in the present study we measured gamma enolase levels in the above mentioned regions of the brain as separate entities, in order to identify the most susceptible regions of the brain to 1-BP. However, we did not observe any significant changes in gamma enolase levels in different brain regions when compared to the corresponding control values. We speculate that dissection of brain into small parts might result in larger variation in measurement.
Workers exposed to 1-BP in North Carolina, USA, described the appearance of a variety of neurological symptoms such as numbness in the legs, headache, stumbling, diarrhea, changes in sweating pattern and urinary incontinence (Ichihara 2005; Ichihara et al., 2002). The measured exposure levels were 60–261 ppm, but the true exposure levels would be higher than those values, because the exposure levels were measured following improvement of ventilation after identification of human cases. The other victims from Utah, USA, did not show complete recovery even several years after exposure to 1-BP, which was estimated to be 130 ppm (range 91-176 ppm) with a time-weighted average of 108 ppm (range 92–127 ppm) (Majersik et al., 2007). It is difficult to directly compare the effects of different exposure levels on humans and animals at this stage, because of possible species difference in susceptibility. The present study showed biphasic response to 1-BP exposure in rats, and accordingly there should be a critical exposure level that affects reversibility and recovery, depending on the toxicological endpoints. The critical level in humans should be investigated in further studies.
In conclusion, the present study revealed that exposure to 1-BP at a high dose depleted spermatogenic cells, similar to 2-BP, in contrast to the effect of a lower dose that caused only spermiation failure. The depletion of spermatogenic cells, which may also explain the reduced testicular weight, epididymal weight and sperm count, lasted longer than other temporary changes such as spermiation failure, decrease in plasma testosterone level, abnormal histopathological changes and weight reduction of seminal vesicle and prostate. The decrease in hindlimb muscle strength was persistent, and it could be due to the effect of 1-BP on the central nervous system, peripheral nervous system or the muscles themselves, which requires further investigation.
|
FUNDING |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Japan Society for the Promotion of Science grant (16390169).
|
|
NOTES |
|---|
This paper was presented in part at the 46th Annual Society of Toxicology Meeting in Charlotte, NC, in March 2007.
|
ACKNOWLEDGMENTS |
|---|
The authors declare they have no competing financial interests.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. (1976) 72:248–254.[CrossRef][Web of Science][Medline]
Harney J, Nemhauser J, Reh C, Trout D, Schrader S. NIOSH Health Hazard evaluation report. In: Sawmills (2003) NC, USA.
Ichihara G. Neuro-reproductive toxicities of 1-bromopropane and 2-bromopropane. Int. Arch. Occup. Environ. Health (2005) 78:79–96.[CrossRef][Web of Science][Medline]
Ichihara G, Asaeda N, Kumazawa T, Tagawa Y, Kamijima M, Yu X, Kondo H, Nakajima T, Kitoh J, Yu IJ. Testicular and hematopoietic toxicity of 2-bromopropane, a substitute for ozone layer-depleting chlorofluorocarbons. J. Occup. Health (1997) 39:57–63.[Web of Science]
Ichihara G, Asaeda N, Kumazawa T, Tagawa Y, Kamijima M, Yu X, Kondo H, Nakajima T, Kitoh J, Yui J. Testicular toxicity of 2-bromopropane. J. Occup. Health (1996) 38:205–206.
Ichihara G, Kitoh J, Yu X, Asaeda N, Iwai H, Kumazawa T, Shibata E, Yamada T, Wang H, Xie Z. 1-Bromopropane, an alternative to ozone layer depleting solvents, is dose-dependently neurotoxic to rats in long-term inhalation exposure. Toxicol. Sci. (2000a) 55:116–123.
Ichihara G, Miller JK, Ziolkowska A, Itohara S, Takeuchi Y. Neurological disorders in three workers exposed to 1-bromopropane. J. Occup. Health (2002) 44:1–7.[CrossRef][Web of Science]
Ichihara G, Yu X, Kitoh J, Asaeda N, Kumazawa T, Iwai H, Shibata E, Yamada T, Wang H, Xie Z. Reproductive toxicity of 1-bromopropane, a newly introduced alternative to ozone layer depleting solvents, in male rats. Toxicol. Sci. (2000b) 54:416–423.
Ichihara S, Yamada Y, Ichihara G, Nakajima T, Li P, Kondo T, Gonzalez FJ, Murohara T. A Role for the aryl hydrocarbon receptor in regulation of ischemia-induced angiogenesis. Arterioscler. Thromb. Vasc. Biol. (2007) 27:1297–1304.
Jamie DG, Sarah TRM, Bernhard HJJ, Benjamin WCR. The effect of glutamine on locomotor performance and skeletal muscle myosins following spinal cord injury in rats. J. Appl. Physiol. (2006) 101:1045–1052.
Kato K, Suzuki F, Umeda Y. Highly sensitive immunoassays for three forms of rat brain enolase. J. Neurochem. (1981) 46:1783–1788.
Kim Y, Jung K, Hwang T, Jung G, Kim H, Park J, Kim J, Park D, Park S, Choi K. Hematopoietic and reproductive hazards of Korean electronic workers exposed to solvents containing 2-bromopropane. Scand. J. Work Environ. Health (1996) 22:387–391.[Web of Science][Medline]
Ku W, Chapin R, Wine R, Gladen B. Testicular toxicity of boric acid (BA): Relationship of dose to lesion development and recovery in the F344 rat. Reprod. Toxicol. (1993) 7:305–319.[CrossRef][Web of Science][Medline]
Lee HS, Kang BH, Son HY, Kim HY, Cho YC, Roh JK. The effects of 2-bromopropane on hematopoiesis and spermatogenesis in Sprague-Dawley male rats. Toxicol. Public Health (1998) 14:129–141.
Leone M, Costa M, Capitanio GL, Palmero S, Prati M, Leone MM. Dibromochloropropane (DBCP) effects on the reproductive function of the adult male rat. Acta Eur. Fertil. (1988) 19:99–103.[Medline]
Majersik JJ, Caravati M, Steffens DJ. Severe neurotoxicity associated with exposure to the solvent 1-bromopropane (n-propyl bromide). Clin. Toxicol. (2007) 45:270–276.[CrossRef][Web of Science]
Meyer O, Tilson H, Byrd W, Riley M. A method for the routine assessment of fore-and hind-limb grip strength of rats and mice. Neurobehav. Toxicol. (1979) 1:233–236.[Medline]
Mori K, Kaido M, Fujishiro K, Inoue N, Koide O, Hori H, Tanaka I. Dose-dependent effects of inhaled ethylene oxide on spermatogenesis in rats. J. Ind. Med. (1991) 48:270–274.
B e. p., Boekelheide K, Darney SP, Daston GP, David RM, Luderer U, Olshan AF, Sanderson WT, Willhite CC, Woskie S, NTP Center, (2004). NTP-CERHR Expert Panel Report on the reproductive and developmental toxicity of 2-bromopropane. Reprod. Toxicol. 18:189–217.
Omura M, Tanaka A, Hirata M, Zhao M, Yuji M, Inoue N, Gotoh K, Ishinishi N. Testicular toxicity of gallium arsenide, indium arsenide, and arsenic oxide in rats by repetitive intratracheal instillation. Fund. Appl. Toxicol. (1996) 32:72–78.[CrossRef][Web of Science][Medline]
Russell LD, Etllin RA, Hikim APS, Clegg ED. Histological and histopathological evaluation of the testis. In: In Histological and Histopathological Evaluation of the Testis (1990) Florida Clearwater, Florida-34620: Cache River Press. 210–264.
Saegusa J. Cumulative effects of 1,2-dibromo-3-chloropropane(DBCP) on kidney and testis. Ind. Health (1989) 27:49–58.[Web of Science][Medline]
Shemi D, Sod-Moriah UA, Kaplanski J, Potashnik G, Yanai-Inbar I. Suppression and recovery of spermatogenesis in dibromo-chloropropane treated rats. Andrologia (1982) 14:191–199.[Web of Science][Medline]
Son HY, Cho SW, Kim YB, Ha CS, Kang BH. Testicular lesion in the Sprague-Dawley rats treated with high dose of 2-bromopropane. Korean J. Vet. Pathol. (1997) 1:97–105.
Son HY, Cho SW, Kim YB, Ha CS, Kang HB. Testicular atrophy induced by 2-bromopropane in the Sprague-Dawley rats. J. Toxicol. Public Health (1998) 14:143–149.
Son HY, Kim YB, Kang BH, Cho SW, Ha CS, Roh JK. Effects of 2-bromopropane on spermatogenesis in the Sprague-Dawley rat. Reprod. Toxicol. (1999) 13:179–187.[CrossRef][Web of Science][Medline]
Takeuchi Y, Huang J, Shibata E, Hisanaga N, Ono Y. A trial to automatize an organic solvent exposure system for small animals. Jpn. J. Ind. Health (1989) 31:722.
Takeuchi Y, Ichihara G, Kamijima M. A review on toxicity of 2-bromopropane: Mainly on it's reproductive toxicity. J. Occup. Health (1997) 39:179–191.[Web of Science]
Wang H, Ichihara G, Ito H, Kato K, Kitoh J, Yamada T, Yu X, Tsuboi S, Moriyama Y, Sakatani R. Biochemical changes in the central nervous system of rats exposed to 1-bromopropane for seven days. Toxicol. Sci. (2002) 67:114–120.
Wang H, Ichihara G, Ito H, Kato K, Kitoh J, Yamada T, Yu X, Tsuboi S, Moriyama Y, Takeuchi Y. Dose-dependent biochemical changes in rat central nervous system after 12-week exposure to 1-bromopropane. Neurotoxicology (2003) 24:199–206.[CrossRef][Web of Science][Medline]
Wenger CB, Roberts MF, Nadel ER, Stolwijk JA. Thermoregulatory control of finger blood flow. J. Appl. Physiol. (1975) 38:1082–1975.
Yu IJ, Chung YH, Lim CH, Maeng SH, Lee JY, Kim HY, Lee SJ, Kim CH, Kim TG, Park JS. Reproductive toxicity of 2-bromopropane in Sprague Dawley rats. Scand. J. Work Environ. Health (1997) 23:281–288.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  F. Liu, S. Ichihara, S. S. Mohideen, U. Sai, J. Kitoh, and G. Ichihara Comparative Study on Susceptibility to 1-Bromopropane in Three Mice Strains Toxicol. Sci., November 1, 2009; 112(1): 100 - 110. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||