Increased Dementia Mortality in West Virginia Counties with Mountaintop Removal Mining?

Atmospheric particulate matter (PM) is elevated in areas of mountaintop removal mining (MTM), a practice that has been ongoing in some counties of West Virginia (WV) USA since the 1970s. PM inhalation has been linked to central nervous system pathophysiology, including cognitive decline and dementia. Here we compared county dementia mortality statistics in MTM vs. non-MTM WV counties over a period spanning 2001-2015. We found significantly elevated age-adjusted vascular or unspecified dementia mortality/100,000 population in WV MTM counties where, after adjusting for socioeconomic variables, dementia mortality was 15.60 (±3.14 Standard Error of the Mean (S.E.M.)) times higher than that of non-MTM counties. Further analyses with satellite imaging data revealed a highly significant positive correlation between the number of distinct mining sites vs. both mean and cumulative vascular and unspecified dementia mortality over the 15 year period. This was in contrast to finding only a weak relationship between dementia mortality rates and the overall square kilometers mined. No effect of living in an MTM county was found for the rate of Alzheimer's type dementia and possible reasons for this are considered. Based on these results, and the current literature, we hypothesize that inhalation of PM associated with MTM contributes to dementia mortality of the vascular or unspecified types. However, limitations inherent in ecological-type studies such as this, preclude definitive extrapolation to individuals in MTM-counties at this time. We hope these findings will inspire follow-up cohort and case-controlled type studies to determine if specific causative factors associated with living near MTM can be identified. Given the need for caregiving and medical support, increased dementia mortality of the magnitude seen here could, unfortunately, place great demands upon MTM county public health resources in the future.

Analysis of extinction acquisition to attenuated tones in prenatally stressed and non-stressed offspring following auditory fear conditioning.

Stimulus generalization occurs when stimuli with characteristics similar to a previously conditioned stimulus (CS) become able to evoke a previously conditioned response. Experimental data (Lissek et al., 2005) indicate that patients with post-traumatic stress disorder (PTSD), more often show stimulus generalization following fear conditioning when tested under laboratory conditions. Factors surrounding this observation may contribute to two common features of PTSD: 1) hyper-responsiveness to sensory stimuli reminiscent of those associated with the original trauma, and 2) resistance of PTSD to extinction-based therapies. Adverse early experience is considered a risk factor for the later development of PTSD and in the present experiments we hypothesized that stimulus generalization would occur in an animal model of adverse early experience, the prenatally stressed (PS) rat. Adult PS and control (CON) rats underwent extensive pre-habituation to a conditioning chamber followed by conventional auditory fear conditioning. The next day both groups began an extinction regimen where a series of quieter (attenuated), CSs were administered prior to the full 75 dB training CS. When tested in this manner, PS rats froze at significantly lower tone amplitudes than did CON offspring on the first day of extinction training. This suggests that the PS rats had stimulus-generalized the CS to lower decibel tones. In addition to this finding, we also observed that PS rats froze more often and longer during three ensuing days of extinction training to attenuated tones. Group differences vanished when PS and CON rats were extinguished under conventional conditions. Thus, it appears that the two extinction regimens differed in their aversive cue saliency for the PS vs. CON rats. Follow-up prefrontal cortex transcriptome probing suggests that cholinergic and dopaminergic alterations may be involved.

Effects of prenatal stress on lever-press acquisition with delayed reinforcement in male and female rats.

The prenatally stressed (PS) rat shows enhanced conditioned fear and increased behavioral inhibition in response to footshock compared to control (CON) rats. It is unclear whether this facilitated learning will occur only with aversive stimulation, or if it will also be observed in the context of positive reinforcement. There are limited and inconsistent data regarding sex differences and the impact of prenatal stress on learning. The present study was designed to examine lever-press acquisition with a 10-s delay to food reinforcement in male and female PS and CON rats. Overall, twice as many PS male rats acquired the lever-press response than the PS female rats, CON male rats, and CON female rats. PS male rats also earned significantly more reinforcers and responded on the operative lever at a significantly greater rate than the other three rat groups. These findings suggest that PS rats exhibit altered learning with a task involving positive reinforcement, and this effect of PS is sex specific for male rats.

Blunted amygdalar anti-inflammatory cytokine effector response to postnatal stress in prenatally stressed rats.

Smad1 is the downstream effector for bone morphogenetic protein, part of the anti-inflammatory cytokine family. Glucocorticoids (GCs) increase the production of anti-inflammatory cytokines to oppose the actions of pro-inflammatory cytokines. Here we used the prenatally stressed (PS) rat to see if chronic GC activation affects this protective mechanism in the amygdala. Male PS and control offspring were either left undisturbed or exposed to a 2-week regimen of intruder stress. One week later, half of these animals were further subjected to restraint stress for 3 days. Nuclear and cytoplasmic phosphorylated (p)-Smad1 were visualized by immunocytochemistry and quantified in the lateral and basolateral amygdala and in the hind limb primary somatosensory (S1HL) cortex. PS rats showed significantly greater baseline p-Smad1 per cell than controls. However, intruder stress increased p-Smad1 nuclear staining in the control rats only: no further increases in either compartment were observed in the PS group. With repeated restraint stress, attenuation of both cytoplasmic and nuclear p-Smad1 responses was significantly greater in controls. Thus, the overall p-Smad1 responsiveness of amygdala neurons of PS rats to life stressors is blunted. We hypothesize that the amygdala may play an essential role in initiating the cytokine response to stress in the adult rat brain. Basal p-Smad1 staining was unaffected by prenatal stress in the S1HL cortex but became elevated in the cytoplasm following intruder stress. The significance of this is unknown, but may point to a means by which stress can generally affect cells whose functions are unrelated to driving the sympathoadrenal system.

Prenatal stress affects the developmental trajectory of the rat amygdala.

The amygdala plays a critical role in generating the emotion of fear, and alterations in amygdala fear processing are thought to underlie the acquisition and maintenance of anxiety disorders. The prenatally stressed (PS) rat displays hormonal, behavioral and brain anatomical similarities to anxious humans and is useful to study the neurobiological underpinnings of pathological anxiety. We studied PS and control male rats at postnatal days 7 (P7), P25, P45 and P60. Using unbiased stereological analyses we examined the volumes, anterior-posterior lengths and total numbers of neurons and glia of the basolateral (BL), central (Ce) and lateral (La) amygdalar nuclei. We found prenatal stress-associated differences in the developmental trajectories of each nucleus. These were apparent in some measures as early as P7, most extensive at P25 and resolved by P45, at least as seen by Nissl staining. These changes were not a result of differential brain growth. This early divergence in developmental trajectories seen here may be the harbinger of PS rat amygdalas that ultimately function very differently in adulthood.

Morphological plasticity in the neural circuitry responsible for seasonal breeding in the ewe.

An increase in the response of GnRH neurons to estrogen negative feedback is responsible for seasonal anestrus in the ewe, but the underlying neural mechanisms remain largely unknown. Neural plasticity may play an important role because the density of synaptic input to GnRH neurons changes with seasons. Moreover, the transition from breeding to anestrous season requires thyroid hormones, which are also required for neuronal development. In the first experiment, we examined whether the decrease in synapses on GnRH neurons is critical for the transition to anestrus by comparing synaptic input in thyroidectomized and thyroid-intact controls, using electron microscopic analysis. Thyroidectomized ewes remained in the breeding season, but the number of synaptic contacts on their GnRH cells was not different from those in thyroid-intact ewes that were anestrus. The next experiment tested whether there was a seasonal change in morphology of the A15 dopaminergic neurons that mediate estrogen negative feedback during anestrus by analyzing synapsin-positive close contacts onto A15 neurons with confocal microscopy. There was a 2-fold increase in these close contacts onto dendrites of A15 neurons in anestrus and a corresponding increase in the length of A15 dendrites at this time of year. The increase in dendritic length was blocked by thyroidectomy, but this procedure did not significantly affect synaptic input to A15 neurons. These results provide initial evidence that the seasonal change in synapses on GnRH neurons is not sufficient for the transition into anestrus but that plasticity of the A15 dopaminergic neurons mediating estrogen negative feedback may contribute to this seasonal alteration.

Low-frequency hearing loss in prenatally stressed rats.

We investigated the possibility that hearing thresholds are altered in prenatally stressed rats raised in a normal auditory environment. Pregnant dams were assigned randomly to prenatally stressed and control groups. Half of the dams were subjected to the mild stressors of handling, exposure to a novel cage and saline injection at random times during lights-on daily. The hearing thresholds of young adult male offspring were assessed by recording auditory-evoked brainstem responses to 0.5, 1, 2, 4, 8, 16, 32 and 64 kHz pure tones. The resultant audiograms showed that prenatally stressed offspring had significantly higher hearing thresholds than control animals at 1, 2 and 4 kHz (t-tests, P<0.05). The threshold shifts caused by prenatal stress averaged 7.7 dB across frequencies. We conclude that prenatal stress causes low-frequency hearing loss, possibly due to increased vulnerability to noise-induced hearing loss, accelerated cochlear degeneration and/or disrupted cochlear development.

Perinatal corticosteroid effect on amygdala and hippocampus volume during brain development in the rat model.

Exposure of the fetus to corticosteroid during brain development has been suggested to cause permanent change in brain structure and has been associated with long term cognitive, behavioral and emotional impairment. We evaluated the effect of perinatal corticosteroid, at a dose similar to that which human fetuses are exposed, on cerebral cortex, corpus collosum, hippocampus, dentate gyrus and amygdala in a rat model. Rat pups were given betamethasone at day 1 (P1). Brain sections from the rat pups at postnatal day 45 (P45) were then analyzed. No differences were noted in the volumes of cerebral cortex, corpus collosum, hippocampus, dentate gyrus, or three nuclei of the amygdala compared to the control and sham groups. We concluded that a single course of betamethasone, at a comparable dose to that which the human fetus is exposed in clinical practice, had no effect on these regional brain volumes at this stage of development.

Early emergence of increased fearful behavior in prenatally stressed rats.

We have been studying the mildly prenatally stressed (PS) rat as a potentially useful animal model of anxiety disorders. Previously we have demonstrated that there are anatomical and biochemical alterations in the amygdalas of adult PS offspring and that these offspring show increased fearful behaviors. However, human data indicate that anxiety disorders often present first in early childhood and then persist throughout adolescence and adulthood. To determine if PS rats also model this characteristic of human anxiety disorders, here we asked whether behavioral indices of increased fear would be detectable at an early age. We tested the hypotheses that young PS rats would show increased behavioral fearfulness in response to an acute stressor and that this would increase with age. A mild prenatal stressor, consisting of removal of the dam from the home cage and administration of a subcutaneous injection of 0.1 ml of 0.9% saline daily, was administered during the last week of pregnancy. Offspring were tested in the defensive-withdrawal apparatus before and after exposure to restraint stress at 25, 45 and 60 days of age. PS animals showed increased defensive-withdrawal behavior following the stressor and were more fearful following restraint when compared to controls (CON). This was significant at P45 and increased to P60. Hence, fearful behaviors in PS rats emerge prior to sexual maturation and increase in magnitude thereafter, further validating our model as a means to investigate the underpinnings of anxiety disorders.

Divergent glial fibrillary acidic protein and its mRNA in the activated supraoptic nucleus.

Previous studies have shown decreased immunoreactive glial fibrillary acidic protein (GFAP) in the supraoptic nucleus (SON) when magnocellular neuroendocrine cells (MNCs) are activated by lactation or dehydration. This is thought to underlie structural plasticity of glial processes that occurs during these times. Here, we investigated how this apparent reduction in protein relates to GFAP mRNA expression in the dehydrated rat as visualized by in situ hybridization. Densitometry of silver grains in the SON revealed low levels of mRNA expression in control, 2-day dehydrated and 21-day rehydrated (R21) animals. Conversely, the SON from 7-day dehydrated (D7) subjects displayed significantly more silver grains. Thus, the pattern of GFAP mRNA expression is the inverse of what we previously observed for GFAP immunoreactivity in tissue sections of the SON. No differences in mRNA levels due to hydration state were seen in the lateral hypothalamic area, suggesting that increases in GFAP mRNA at D7 were specifically related to MNC activation. These data indicate a divergence in GFAP mRNA and protein expression in the SON.

Glial limitans elasticity subjacent to the supraoptic nucleus.

Two previous studies from our laboratory have indicated that the ventral glial limitans subjacent to the hypothalamic supraoptic nucleus (SON-VGL) undergoes a reversible thinning upon chronic activation of the magnocellular neuroendocrine cells (MNCs) of the supraoptic nucleus (SON). Numerous other studies have shown that MNC somata hypertrophy with activation. One aim of the current study was to understand better how SON-VGL thinning occurs. A second aim was to quantify overall changes of the MNC somata region due to cellular hypertrophy to compare relative changes in dimensions. Here, we undertook a light microscopic stereological investigation of the SON and the subjacent SON-VGL of Nissl stained material under basal and activated conditions. Astrocyte numbers in the underlying SON-VGL remained stable across hydration state as did the overall volume of the SON-VGL and dendritic zone reference area. How these data are consistent with our earlier observations of SON-VGL thinning was resolved by the finding of a highly significant, 30% increase in the mediolateral dimension of the SON-VGL in dehydrated rats. These observations fit well with previous work from our laboratory that demonstrates a reorientation of SON-VGL astrocytes, from vertical to horizontal, which occurs in the activated SON-VGL. We found a significant, approximately 54%, increase in the overall volume of the MNC region of the SON. No significant rostrocaudal lengthening of the SON was detected, although a trend was evident. All the observed changes reversed with rehydration. These data indicate that elasticity of the SON-VGL acts to accommodate the volume expansion of the MNCs and enables the SON-VGL to continue as an interface between the underlying cerebrospinal fluid in the subarachnoid space and the expanded SON above.

Lateral amygdaloid nucleus expansion in adult rats is associated with exposure to prenatal stress.

Anxiety disorders in humans have been associated with chronic activation of the hypothalamic-pituitary-adrenal axis and changes in the volume of the amygdala. Interest in the etiology of anxiety disorders has led us and others to investigate the effects of prenatal stress on the brain development of adult male rat offspring. Prenatally stressed rats represent a promising animal model for anxiety disorders in that they have already been characterized as having both upregulated corticotropin-releasing factor (CRF) brain biochemistry and altered, more fearful, behaviors. Consistent with this, there is now evidence that prenatal stress also has an impact on the development of CRFergic neurons in the hypothalamic paraventricular nucleus and neurogenesis in the hippocampus. At this time, little information about the impact of prenatal stress on amygdala anatomy has been presented. Here we asked whether prenatal stress also has an impact on the development of the amygdala, because this structure plays a direct role in the emotions of anxiety and fear. Stereological measures of well-defined subregions of amydgdaloid nuclei revealed significantly expanded dimensions of the lateral nucleus in prenatally stressed offspring, due, in part, to more neurons and glia. These data may have direct import for the effect of adverse early life experiences and the etiology of anxiety disorders in humans. They also imply that early experiences may not be "grown out of" with development; in fact, the opposite might be true-adverse early life experiences may set developmental events into motion in the brain that last a lifetime.

Increased morphological diversity of microglia in the activated hypothalamic supraoptic nucleus.

Microglia are the immune cells of the CNS. In the normal adult mammalian brain, the majority of these cells is quiescent and exhibits a ramified morphology. Microglia are perhaps best known for their swift transformation to an activated ameboid morphology in response to pathological insults. Here we have observed the responsiveness of these cells to events surrounding the normal activation of neurosecretory neurons in the hypothalamic supraoptic nucleus (SON), a well studied model of structural plasticity in the CNS. Neurons in the SON were activated by substituting 2% saline for drinking water. Brain sections were collected from four experimental groups [controls (C), 2 d-dehydrated (2D), 7 d-dehydrated (D7), and 7 d-dehydrated/21 d-rehydrated animals (R21)] and stained with Isolectin-B4-HRP to visualize microglial cells. Based on morphological criteria, we quantified ramified, hypertrophied, and ameboid microglia using unbiased stereological techniques. Statistical analyses showed significant increases in the number of hypertrophied microglia in the D2 and D7 groups. Moreover, there was a significant increase in the number of ameboid microglia in the D7 group. No changes were seen across conditions in the number of ramified cells, nor did we observe any significant phenotypic changes in a control area of the cingulate gyrus. Hence, increased morphological diversity of microglia was found specifically in the SON and was reversible with the cessation of stimulation. These results indicate that phenotypic plasticity of microglia may be a feature of the normal structural remodeling that accompanies neuronal activation in addition to the activation that accompanies brain pathology.

Mild prenatal stress in rats is associated with enhanced conditioned fear.

We tested the hypothesis that prenatal stress would enhance conditioned fear in adult rats. Pregnant Sprague-Dawley rats were stressed by exposure to a novel environment and subcutaneous injection of saline (0.1 ml 0.9% NaCl) at random times daily from Days 14 to 21 of pregnancy. When compared to adult control (CON) male rats from unmanipulated pregnancies, adult prenatally stressed (PS) male rats showed increased freezing behavior in response to acute footshock as well as increased freezing behavior the next day in the same context, without shock delivery. In another experiment, the gestational stressor was examined for elevations in corticosterone and ACTH. At gestational days (G)15, G17, G19 and G21, maternal and fetal plasma was collected. Analysis showed elevations in corticosterone and ACTH in the PS dams when compared to the CON dams. Additionally, increased corticosterone was found in the PS fetuses when compared to the CON fetuses. Finally, some CON and PS litters were examined for alterations in length of gestation, number of pups born, bodyweight on postnatal day (P)1 and anogenital distance on P1 and differences were not found. In conclusion, our data demonstrate that a mild stressor during gestation, sufficient to raise plasma corticosterone and ACTH, is associated with enhanced conditioned fear during adulthood.

Effects of prenatal stress on defensive withdrawal behavior and corticotropin releasing factor systems in rat brain.

Exposure of pregnant rats to stress results in offspring that exhibit abnormally fearful behavior and have elevated neuroendocrine responses to novelty and aversive stimuli. This study examined the effects of prenatal stress on plasma corticosterone, adrenal weight, defensive withdrawal behavior, and the density of receptors for corticotropin releasing factor (CRF) in the amygdala. Pregnant Sprague-Dawley rats were stressed by daily handling and saline injection (s.c., 0.9%, 0.1 mL) during the last week of gestation. Male offspring were studied at adulthood (60-120 days of age). Adrenal hypertrophy and increased plasma corticosterone were observed in the prenatally stressed offspring. Defensive withdrawal, an ethological measure of the conflict between exploratory behavior and retreat, was quantified in naive offspring, and in offspring exposed to restraint stress (2 h). Restraint stress increased defensive withdrawal in both control and prenatally stressed offspring. Both naive and restraint-stressed prenatally stressed offspring exhibited increased defensive withdrawal compared to control offspring. There was a significant interaction between prenatal stress and restraint stress, suggesting increased vulnerability of prenatally stressed offspring. The effects of restraint in the defensive withdrawal test were reduced by intracerebroventricular administration of the CRF antagonists, alpha-helical CRF9-41 (20 microg every hour) or D-phe(12), Nle(21, 38), C(alpha)-MeLeu(37)]-CRF((12-41)) (5 microg every hour) during the restraint period. The difference between control and prenatally stressed offspring was abolished by the CRF antagonists, suggesting that increased activation of CRF receptors may be a factor in the behavioral abnormalities of prenatally stressed rats. Measurement of CRF receptors in amygdala revealed a 2.5-fold increase in binding in prenatally stressed offspring. In light of previous work from this laboratory demonstrating increased content and release of CRF in amygdala from prenatally stressed offspring, the present study suggests that the increased fearfulness of prenatally stressed rats may be a consequence of increased activity of CRFergic systems in the amygdala.

Mechanisms of glial retraction in the hypothalamo-neurohypophysial system of the rat.

It has been known for more than twenty years that changes in glial coverage of magnocellular neurones in the hypothalamo-neurohypophysial system accompany activation of those neurones. This led to the so-called 'glial retraction hypothesis.' However, until recently, little has been established as to how this structural plasticity of astrocytes develops. This paper will explore a number of hypotheses and supporting data concerning these changes.

Dehydration-associated changes in the ventral glial limitans subjacent to the supraoptic nucleus include a reduction in the extent of the basal lamina but not astrocytic process shrinkage.

In these studies we have investigated factors that might account for two previous observations of the ventral glial limitans subjacent to the supraoptic nucleus (SON-VGL) of dehydrated rats: (1) a reversible reduction in the thickness of the SON-VGL, and (2) a reversible reorientation of VGL astrocytes. Since components of the basal lamina influence both cell viability and polarity, we used electron microscopic sterology to determine the volume fraction of basal lamina in the SON-VGL. We further made extensive measurements of astrocytic process thickness to determine if cellular shrinkage is a factor in the thinning of the SON-VGL. While we found no evidence for changes in the thickness of astrocytic processes, there was a significant and reversible reduction in the extent of the basal lamina. These data suggest that the thinning of the VGL is due to complex biochemical events and is not merely an epiphenomenon of dehydration.

The surface density of glial fibrillary acidic protein immunopositive astrocytic processes in the rat supraoptic nucleus is reversibly altered by dehydration and rehydration.

Ultrastructural and immunohistochemical studies of the supraoptic nucleus (SON) have provided evidence that retraction and extension of astrocytic processes from between magnocellular neuroendocrine cells (MNCs) likely plays a role in the release of oxytocin, and/or vasopressin, that accompanies parturition, lactation and dehydration. The present study estimates the surface density (Sv) of glial fibrillary acidic protein (GFAP)-immunoreactivity, predominantly in astrocytic processes, in the SON of normally hydrated, dehydrated and rehydrated rats. The Sv of GFAP processes in dehydrated rats was significantly reduced compared with control levels. Rehydration returned Sv to control levels. The reversible reduction in Sv indicates that the previously observed reduction in optical density is due to a rearrangement of astrocyte processes in the SON which occurs at the same time as the selective functional activation of MNCs.

Dehydration and rehydration selectively and reversibly alter glial fibrillary acidic protein immunoreactivity in the rat supraoptic nucleus and subjacent glial limitans.

Ultrastructural studies of the supraoptic nucleus (SON) of the hypothalamus suggest that an active retraction and extension of astrocytic processes (structural plasticity) from between magnocellular neuroendocrine neurons plays a role in the release of oxytocin, vasopressin, or both peptides that accompanies parturition, lactation, and dehydration. In support of this, Salm et al. (1985) previously demonstrated a lactation-associated reduction in immunoreactive glial fibrillary acidic protein (GFAP), an astrocyte-specific cytoskeletal constituent. To determine if similar changes occur in response to dehydration, and if they are reversible, the present study examined GFAP-immunoreactivity (IR) in the SON under various hydration states. Rats were dehydrated for 7 days by substitution of drinking water with 2% saline (n = 3), or dehydrated for 7 days followed by 7 days of rehydration (n = 3). A control group (n = 3) with free access to tap water was used for comparisons. The optical density of GFAP-IR was obtained from the SON, globus pallidus, and lateral hypothalamic regions. The areas of the ventral glial limitans subjacent to the SON (SON-VGL) and of linearly equivalent segments of glial limitans more distant from the SON were also determined. Dehydration resulted in a significant reduction in GFAP-IR in the SON compared to control and rehydrated levels. We also found that the area of the SON-VGL was significantly larger than that of linearly equivalent segments of glial limitans elsewhere and that it was significantly reduced in dehydrated rats, returning to control levels with rehydration. GFAP-IR and glial limitans thickness in regions unrelated to body fluid homeostasis lateral to the SON, overlying to dorsal cortex, and subjacent to the optic chiasm were not significantly changed by hydration state. These results are similar to the changes of GFAP-IR reported for lactating rats and provide further evidence for a role of structural plasticity of astrocytes in events surrounding the selective functional activation of local neurons.

Glutathione S-transferase π-isoform is not altered in bladder tissue from smokers.

Glutathione S-transferase π (GSTπ)-isoform is a cytosolic enzyme involved in the detoxification of reactive metabolites generated from environmental pollutants and cigarette smoke. Although cigarette smoking has been implicated in the etiology of bladder cancer, no information yet exists regarding GSTπ in smokers and nonsmokers. This study was carried out to evaluate the immunohistochemical localization and to measure levels of immunoreactive GSTπ in bladder tissue from smokers and nonsmokers. Tissues from patients diagnosed with transitional cell carcinoma (TCC) were obtained from paraffin embedded blocks fixed in formalin. Bladder tissues from smokers (n = 7) and nonsmokers (n = 8), and histologically confirmed cancerous and noncancerous areas of the same patients (n = 10) were studied. The immunoreactive GSTπ was quantified by calculating its optical density with a computerized Olympus BH-2 microscope connected to a DAGE camera. Immunohistochemical staining for GSTπ appeared to be evenly distributed in the cytosol of the transitional epithelium (TE) of the noncancerous regions. In the TE from patients with advanced TCC, the staining intensity appeared to be stronger in the nuclei relative to cytoplasm, an effect that was even more pronounced in poorly differentiated cancers and in cancers with squamoid features. The immunoreactive levels of GSTπ in the superficial TE cells was approximately 1.7-fold higher compared with the rest of the TE layer (p < 0.05) in smokers and nonsmokers. No significant differences (p > 0.05) were observed between smokers and nonsmokers in either of these regions. The higher concentration of GSTπ in the TE is suggestive of the protective role of this enzyme, serving to prevent any potentially harmful xenobiotics from entering the bladder tissue. The lack of differences in the detoxifying enzymes between smokers and nonsmokers suggests that the increased susceptibility of bladder cancer in smokers is probably mediated by other mechanisms.