Prenatal smoke exposure: effects on infant auditory system and placental gene expression.

Prenatal smoke exposure has been shown to change cochlear echo response amplitudes and auditory brainstem response (ABR) wave latencies in newborns. Since gene expression changes are often synchronized in different tissue types, the goal of the present work was to determine the relationships between prenatal smoke exposure induced changes in hearing responses with changes in placental gene expression. Results showed significant cotinine level elevations in mothers who smoked ≥10cigarettes/day during their pregnancy compared to no detectable cotinine in nonsmoking mothers. Cochlear echo response amplitudes in the 2-8kHz range and ABR wave latencies, specifically wave V and interpeak interval I-V, were also significantly reduced in newborns of smoking mothers. Functional pathway analysis of upregulated placental genes using the Database for Annotation, Visualization and Integrated Discovery (DAVID) online software showed significant enrichment of terms associated with neurodevelopmental processes including glutamatergic and cholinergic systems and a number of wingless type proteins in the top two tiers with corrected enrichment p-values of ≤0.05. Other relevant functional pathways were significant at unadjusted enrichment p-values of 0.001-0.11 and included calcium signaling, neurotransmission/neurological processes and oxidative stress. The neurological process clusters included 7 genes (EML2, OTOR, SLC26A5, TBL1X, TECTA, USH1C and USH1G) known to modulate cochlear outer hair cell motility. We localized proteins encoded by the top two regulated genes, TBL1X and USH1C, using immunohistochemistry to placental stem and anchoring villi associated with active contractile function. These placental genes may mediate active contraction and relaxation in the placental villi, for example, during maternal-fetal perfusion matching, similar to the active lengthening and shortening of the cochlear outer hair cells during sensory transduction. Thus, the functional consequence of their alteration in the cochlea would be reflected as a decline in cochlear echoes as shown in this study. Such parallel changes suggest the potential utility of placental gene expression as a surrogate for evaluating changes in the developing cochlea related to potential aberrant cochlear function in newborns with prenatal smoke exposure.

Exposure to atrazine affects the expression of key genes in metabolic pathways integral to energy homeostasis in Xenopus laevis tadpoles.

In our laboratory, Xenopus laevis tadpoles exposed throughout development to 200 or 400 µg/L atrazine, concentrations reported to periodically occur in puddles, vernal ponds and runoff soon after application, were smaller and had smaller fat bodies (the tadpole's lipid storage organ) than controls. It was hypothesized that these changes were due to atrazine-related perturbations of energy homeostasis. To investigate this hypothesis, selected metabolic responses to exposure at the transcriptional and biochemical levels in atrazine-exposed tadpoles were measured. DNA microarray technology was used to determine which metabolic pathways were affected after developmental exposure to 400 µg/L atrazine. From these data, genes representative of the affected pathways were selected for assay using quantitative real time polymerase chain reaction (qRT-PCR) to measure changes in expression during a 2-week exposure to 400 µg/L. Finally, ATP levels were measured from tadpoles both early in and at termination of exposure to 200 and 400 µg/L. Microarray analysis revealed significant differential gene expression in metabolic pathways involved with energy homeostasis. Pathways with increased transcription were associated with the conversion of lipids and proteins into energy. Pathways with decreased transcription were associated with carbohydrate metabolism, fat storage, and protein synthesis. Using qRT-PCR, changes in gene expression indicative of an early stress response to atrazine were noted. Exposed tadpoles had significant decreases in acyl-CoA dehydrogenase (AD) and glucocorticoid receptor protein (GR) mRNA after 24 h of exposure, and near-significant (p=0.07) increases in peroxisome proliferator-activated receptor ß (PPAR-ß) mRNA by 72 h. Decreases in AD suggested decreases in fatty acid ß-oxidation while decreases in GR may have been a receptor desensitization response to a glucocorticoid surge. Involvement of PPAR-ß, an energy homeostasis regulatory molecule, also suggested changes in energy status. Despite, or possibly because of, these early gene changes, there were no differences in either absolute ATP levels or ADP:ATP ratios early in the exposure. However, livers from animals exposed to 200 µg/L atrazine had near-significant (p=0.06) increases in ADP:ATP ratios at the end of exposure suggesting tadpoles may have had difficulty maintaining energy homeostasis. Perturbations in the expression of genes regulating energy metabolism by 24 h into exposure to 400 µg/L atrazine was noteworthy, especially since these tadpoles were significantly smaller than controls by 72 h of exposure.

Atrazine exposure affects growth, body condition and liver health in Xenopus laevis tadpoles.

Six studies were performed regarding the effects of atrazine, the most frequently detected pesticide in fresh water in the US, on developing Xenopus laevis tadpoles exposed 5 days post-hatch through Nieuwkoop Faber Stage 62. The levels of atrazine tested included those potentially found in puddles, vernal ponds and runoff soon after application (200 and 400 µg/L) and a low level studied by a number of other investigators (25 µg/L). One study tested 0, 25 and 200 µg/L, another tested 0, 200 and 400 µg/L, while the remaining four studies tested 0 and 400 µg/L. During all exposures, mortality, growth, metamorphosis, sex ratio, fat body (a lipid storage organ) size and liver weights, both relative to body weight, were evaluated. In selected studies, feeding behavior was recorded, livers and fat bodies were histologically evaluated, liver glycogen and lipid content were determined by image analysis, and immunohistochemical detection of activated caspase-3 in hepatocytes was performed. The NOEC was 25 µg/L. None of these exposure levels changed sex ratios nor were intersex gonads noted, however, no definitive histological evaluation of the gonads was performed. Although a marginal increase in mortality at the 200 µg/L level was noted, this was not statistically significant. Nor was there an increase in mortality at 400 µg/L versus controls. At the 400 µg/L level, tadpoles were smaller than controls by 72 h of exposure and remained smaller throughout the entire exposure. Appetite was not decreased at any exposure level. Slowed metamorphosis was noted only at 400 µg/L in two of five studies. Livers were significantly smaller in the study that tested both 200 and 400 µg/L, yet no pathological changes or differences in glycogen or lipid stores were noted. However, livers from 400 µg/L exposed tadpoles had higher numbers of activated caspase-3 immunopositive cells suggesting increased rates of apoptosis. Fat body size decreased significantly after exposure to 200 and 400 µg/L although these organs still contained some lipid and lacked any pathology. Since this was noted across all studies, it was considered the most sensitive indicator of atrazine exposure measured. The changes noted in body and organ size at 200 and 400 µg/L atrazine indicated exposure throughout development compromised the tadpoles. Significant reductions in fat body size could potentially decrease their ability to survive the stresses of metamorphosis or reduce reproductive fitness as frogs rely on stored lipids for these processes.

Chronic exposure to high levels of atrazine alters expression of genes that regulate immune and growth-related functions in developing Xenopus laevis tadpoles.

Atrazine is the most commonly detected pesticide in ground and surface waters, with seasonal spikes that often exceed the Environmental Protection Agency's "Recommended Water Quality Criterion" of 350 parts per billion (ppb). Although numerous studies have shown atrazine produces adverse effects on growth, development, immune and endocrine system functions in a wide range of species, few describe gene expression changes concurrent with atrazine-induced changes in phenotype during development. In this report, developing Xenopus laevis tadpoles were chronically exposed to 400 ppb atrazine, an environmentally relevant concentration. Affymetrix microarrays and Taqman qRT-PCR were used to define gene expression changes that underlie atrazine-induced phenotypic alterations. Atrazine significantly reduced survival and growth (weight, length and fat body size) in male and female tadpoles. Microarray analysis showed atrazine altered expression of 44 genes in male tadpoles (18 upregulated, 26 downregulated) and 77 genes in female tadpoles (23 upregulated, 54 downregulated). Classification of the genes into functional groups showed the majority of genes were associated with the following biological functions: growth and metabolism, proteolysis, fibrinogen complex formation and immune regulation. Seven genes associated with immune system function, specifically defense molecules present in the skin (e.g. magainin II, levitide A, preprocarulein, skin granule protein), were significantly downregulated in female tadpoles. These results support the idea that environmental contaminants such as atrazine compromise important gene pathways during frog development that may, ultimately, be relevant to global amphibian decline.

Effects of polychlorinated biphenyls (PCBs) on expression of neurotrophic factors in C6 glial cells in culture.

Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that affect nervous system function. Glial cells are among the first lines of defense in the nervous system and are involved in activities, including production of neurotrophic factors, which maintain an environment optimally suited for neuronal function. In this study, we investigated the effects of a commercial mixture of PCBs, Aroclor 1254 (A1254), on neurotrophic factor secretion by C6 cells in culture. C6 cells were exposed to medium containing 10 ppm A1254, 0.1% dimethyl sulfoxide (DMSO=vehicle), or normal culture medium. Glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) protein were measured by enzyme-linked immunosorbant assay. GDNF mRNA was measured by real-time RT-PCR. The role of protein kinase C (PKC) signaling in A1254 effects was investigated using bisindolylmaleimide, a PKC antagonist. Exposure to A1254 increased NGF (8.8x10(-5)+/-8.2x10(-6)pg NGF/cell) and GDNF (1.0x10(-4)+/-6.7x10(-6)pg GDNF/cell) secretion compared to DMSO treated controls (5.0x10(-5)+/-7.5x10(-6)pg NGF/cell and 6.2x10(-5)+/-3.1x10(-6)pg GDNF/cell). The effect of A1254 was long-lived, as GDNF secretion was elevated following 5 days of exposure (4.1x10(-5)+/-1.7x10(-6)pg GDNF/cell in A1254 exposed cells vs. 2.9x10(-5)+/-2.3x10(-6)pg GDNF/cell in DMSO exposed cells). GDNF mRNA expression was also elevated following exposure to A1254 (1.14+/-0.07 gene expression units in A1254 exposed cells vs. 0.8+/-0.07 gene expression units in DMSO exposed cells). Bisindolylmaleimide was able to block the effects of A1254 on GDNF secretion. Thus, one potential mechanism by which PCBs may alter nervous system function is via disruption of neurotrophic factor expression by glial cells. The observation that neurotrophic factor expression was increased following exposure to PCB may suggest that glial cells increase expression of neuroprotective genes following exposure to potentially damaging agents such as PCBs.

Gene expression changes in postmortem tissue from the rostral pons of multiple system atrophy patients.

Multiple system atrophy (MSA) is a neurodegenerative disease characterized by various degrees of Parkinsonism, cerebellar ataxia, and autonomic dysfunction. In this report, Affymetrix DNA microarrays were used to measure changes in gene expression in the rostral pons, an area that undergoes extensive damage in MSA, but not other synucleinopathies. Significant changes in expression of 254 genes (180 downregulated and 74 upregulated) occurred in pons tissue from MSA patients when compared with control patients. The downregulated genes were primarily associated with biological functions known to be impaired in Parkinson's disease (PD) and other neurological diseases; for example, downregulation occurred in genes associated with mitochondrial function, ubiquitin-proteasome function, protein modification, glycolysis/metabolism, and ion transport. On the other hand, upregulated genes were associated with transcription/RNA modification, inflammation, immune system function, and oligodendrocyte maintenance and function. Immunocytochemistry, in conjunction with quantitative image analysis, was carried out to characterize alpha-synuclein protein expression as glial cytoplasmic inclusions in the pontocerebellar tract in rostral pons tissue and to determine the relationship between the amount of aggregated alpha-synuclein protein and changes in specific gene expression. Of the regulated genes, 86 were associated with the amount of observed aggregated alpha-synuclein protein in the rostral pons tissue. These data indicate that cells in the pons of MSA patients show changes in gene expression previously associated with the substantia nigra of PD patients and/or other neurological diseases, with additional changes, for example related to oligodendrocyte function unique to MSA.

Aroclor 1254 impairs the hearing ability of Xenopus laevis.

In this study we assessed the effects of chronic, dietary exposure of Aroclor 1254 (A1254) on the hearing of Xenopus frogs. We used the auditory brainstem response (ABR) to assay changes in hearing physiology; ABR thresholds, as well as latency-intensity and amplitude-intensity profiles of the initial positive (P1) and negative (N1) peaks were measured. Two groups of animals that received 50 ppm and 100 ppm of A1254 in their diet from 5 days post-fertilization through metamorphosis were compared to a control group that received untreated chow. The results showed significant threshold elevations in the 3-4 kHz range and significantly delayed peak latencies and reduced amplitudes at these frequencies in A1254 treated animals as compared to control animals. These findings indicate that A1254 selectively damages the high-frequency sensorineural hearing system associated with the basilar papilla of frogs. This preferential damage may be related to inherent differences in the vulnerability of the basilar versus amphibian papilla in the frog. The overall results of this study are also consistent with the reported A1254-induced auditory deficits in mammals indicating that the basilar papilla of the Xenopus frog may serve as an effective model for studying the effects of A1254 on the auditory system.

Polychlorinated biphenyl exposure delays metamorphosis and alters thyroid hormone system gene expression in developing Xenopus laevis.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that disrupt thyroid hormone (TH) system function in numerous species. Previous studies have shown delayed metamorphosis in developing Xenopus laevis frogs exposed to PCBs, but the underlying molecular mechanisms have not been thoroughly investigated. In this research, developing X. laevis tadpoles were exposed to environmentally relevant concentrations (5, 50ppb) of Aroclor 1254 (A1254), a PCB mixture, dissolved in water and 0.25% dimethyl sulfoxide. Quantitative real-time reverse transcriptase polymerase chain reaction was used to measure expression of several TH system genes, other genes that regulate growth and development, and a xenobiotic response gene. Exposure to 50ppb A1254 significantly delayed metamorphosis and significantly altered gene expression of three thyroid system genes: transthyretin and types II and III deiodinase. Since all three genes regulate the amount of available, biologically active TH, PCB-induced changes in the expression of these genes may underlie alterations in metamorphic timing.

Auditory brainstem responses to airborne sounds in the aquatic frog Xenopus laevis: correlation with middle ear characteristics.

In this study we recorded auditory brainstem responses to airborne sounds to determine the hearing sensitivity of Xenopus laevis frogs and correlated their hearing profiles with middle ear characteristics. In newly metamorphosed frogs (body mass 0.5-0.76 gm, snout-vent length 17-20 mm) best hearing sensitivities were measured in the 2.4-2.8 kHz range, whereas optimal hearing sensitivity of older adults (body mass 18-90 gm; snout-vent length 57-100 mm) ranged from 1.0 to 1.2 kHz. Middle ear volumes reconstructed from serial sections showed approximate volume of 0.002 cc and 0.04-0.07 cc in newly metamorphosed and older frogs, respectively. This inverse frequency-volume relationship is consistent with the properties of an acoustic resonator indicating that differences in best hearing sensitivity are at least in part correlated to variation in middle ear volumes for airborne sounds. These results are consistent with peak frequency vibrational velocity profiles of Xenopus tympanic disk that have been shown to be dependent on underlying middle ear volumes and corroborate the occurrence of peak amplitudes of otoacoustic emissions in the 1.0-1.2 kHz region in adult Xenopus frogs.

Dietary exposure to Aroclor 1254 alters gene expression in Xenopus laevis frogs.

Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that contribute to worldwide health problems. Despite data associating PCBs with adverse health effects, decisions to clean up contaminated sites remain controversial. Cleanup decisions are typically based on risk assessment methods that are not sensitive enough to detect subtle changes in health. We have recently shown that gene expression signatures can serve as sensitive molecular biomarkers of exposure and related health effects. Our initial studies were carried out with developing Xenopus laevis tadpoles that were exposed to the PCB mixture Aroclor 1254 (A1254) for 2 days. A1254 was dissolved in dimethyl sulfoxide and added to the aquarium water for rapid loading of PCBs into the tadpole tissue. These studies showed that increases in the expression of specific genes occurred independent of adverse health effects, and decreases in specific genes correlated with the appearance of observable health effects, including decreased survival and gross morphological and behavioral abnormalities. In this report, we extend our previous work to test the use of gene expression signatures as biomarkers in frogs exposed to PCBs through the diet from early tadpole stages through metamorphosis. This work showed that chronic low-dose exposure to A1254 (24 ppm) in food produced tissue levels of 17 ppm and increased gene expression of nerve growth factor and proopiomelanocortin independent of adverse health effects. Exposure to higher doses of A1254 (200 ppm) produced tissue levels of 80 ppm and increased expression of p450 1A1, also, independent of adverse health effects. This work provides further evidence for the use of gene expression changes as biomarkers of exposure to PCBs.

Connexin 43 expression in glial cells of developing rhombomeres of Xenopus laevis.

Connexin 43 is a gap junctional protein found predominantly in astrocytes. In the mammalian nervous system, it appears to play an organizational role during neural development. In the current study, conducted on the frog, Xenopus laevis, we found that connexin 43 occurs in glial cells during development of rhombomeres and that its expression is spatially and temporally regulated. We used neural (2G9) and cell proliferation (BrdU) markers to identify the overall organization of Xenopus rhombomeres and then tracked expression of connexin 43 and glial fibrillary acidic protein, an intermediate filament protein known to mark glia during rhombomeric development. 2G9 was expressed in rhombomeric centers (ventricular concavities) and outlying neuropil regions, whereas BrdU-labeled cells marked boundary regions (ventricular convexities), as early as stage 35/36. These labeling patterns persisted through premetamorphic stages of hindbrain development. At stage 47, 2G9-labeled profiles were highlighted by the presence of connexin 43, and at stage 49/50, connexin 43-labeled profiles, i.e., rhombomeric centers and neuropil, as well as rhombomeric boundaries, not labeled by connexin 43, became immunoreactive to glial fibrillary acidic protein. Cells of rhombomeric center regions and their processes in the outlying neuropil co-expressed glial fibrillary acidic protein and connexin 43 at a time that is characterized by the emergence of hindbrain auditory neural circuitry. Glial fibrillary acidic protein positive glial cells that appeared at rhombomeric boundaries never expressed connexin 43, but rather appeared to physically bisect ventricular convexities into adjacent rhombomeric regions. Thus, glial cells that express connexin 43 in developing rhombomeric centers may be similar to radial glia, assisting in formation of neural circuitry, while glial cells that do not express connexin 43, situated at rhombomeric boundaries, may be involved in demarcating adjacent rhombomeres.

Gene expression patterns predict exposure to PCBs in developing Xenopus laevis tadpoles.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that pose global ecological and human health problems. Although it is well established that PCBs are associated with a variety of adverse health effects in wildlife and in humans, it is often difficult to determine direct cause-and-effect relationships between exposure and specific health outcomes. In this study, gene expression signatures were used to relate exposure to PCBs with altered physiological responses and/or specific health effects. Real-time PCR was used to measure gene expression levels for 10 genes in Xenopus laevis tadpoles (18 days postfertilization, PF) after acute exposure (2 days) to the PCB mixture Aroclor 1254. Specific gene expression signatures correlated with exposure and were predictive of adverse health effects. Exposure to low levels of Aroclor 1254 (5-50 ppb) significantly increased expression of six genes, independent of any health effects; exposure to midlevel concentrations (300-400 ppb) significantly decreased expression levels of two genes, NGF and beta-actin, prior to the onset of observable health effects; exposure to higher doses (500-700 ppb) significantly decreased NGF and beta-actin expression concomitant with the appearance of gross morphological abnormalities, behavioral deficits, and a statistically significant decrease in survival. This study expands upon our previous work that demonstrated an age-dependent susceptibility to Aroclor 1254 in Xenopus laevis tadpoles and that defined specific gene expression signatures as useful bioindicators of exposure and as predictors of overt or impending health effects.

Exposure to the polychlorinated biphenyl mixture Aroclor 1254 alters melanocyte and tail muscle morphology in developing Xenopus laevis tadpoles.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that have damaging effects on both ecosystem and human health. Numerous studies have shown that exposure to PCBs can alter growth and development of aquatic organisms, including frogs. In this report, developing Xenopus laevis tadpoles were exposed to the PCB mixture Aroclor 1254. Tadpoles were exposed from 5 through 9 d postfertilization to either 0, 1, 10, 50, or 100 ppm Aroclor 1254. Exposure to an acute, high concentration of Aroclor 1254 (10, 50, and 100 ppm) caused statistically significant reductions in survival and body size. In addition, tadpoles exposed to these higher concentrations showed histological abnormalities, including aberrant tail tip, myotomal, and melanocyte morphologies. Described adverse health effects associated with PCB exposure of developing frogs will serve as useful health endpoints in ongoing and future molecular-based studies that correlate health effects with changes in gene expression.

Aroclor 1254 alters morphology, survival, and gene expression in Xenopus laevis tadpoles.

PCBs are persistent environmental contaminants that cause a variety of adverse health effects in wildlife and humans. This article describes the use of signature gene expression patterns that link increased PCB exposure with progressive, adverse biological effects. Developing Xenopus laevis tadpoles of two age classes were exposed to the PCB mixture Aroclor 1254 for 2 days. Real-time PCR was used to quantitate mRNA expression for 11 physiologically relevant, potential bioindicator genes. Younger tadpoles (5 days postfertilization) were resistant to Aroclor 1254 and showed few changes in gross morphology, swimming behavior, survival, or gene expression. Older tadpoles (11 days postfertilization) were more susceptible to Aroclor 1254. Exposure to 25 and 50 ppm Aroclor 1254 caused alterations in gross morphology and swimming behavior and statistically significant decreases in survival. These tadpoles showed statistically significant decreases in gene expression for 9 out of the 11 genes measured. Tadpoles exposed to 10 ppm showed incipient health changes but had gene expression profiles similar to the tadpoles treated with higher doses of Aroclor 1254. Tadpoles exposed to 1 ppm did not exhibit any observable adverse health effects, yet statistically significant decreases in gene expression occurred in these tadpoles (4 out of 11 genes). After prolonged exposure, tadpoles exposed to 1 and 10 ppm Aroclor 1254 exhibited health effects similar to those exposed to higher concentrations. Therefore, changes in expression of specific genes may serve not only as molecular bioindicators of Aroclor 1254 exposure but also as predictors of impending adverse health effects.