Compensatory changes in CYP expression in three different toxicology mouse models: CAR-null, Cyp3a-null, and Cyp2b9/10/13-null mice.

Targeted mutant models are common in mechanistic toxicology experiments investigating the absorption, metabolism, distribution, or elimination (ADME) of chemicals from individuals. Key models include those for xenosensing transcription factors and cytochrome P450s (CYP). Here we investigated changes in transcript levels, protein expression, and steroid hydroxylation of several xenobiotic detoxifying CYPs in constitutive androstane receptor (CAR)-null and two CYP-null mouse models that have subfamily members regulated by CAR; the Cyp3a-null and a newly described Cyp2b9/10/13-null mouse model. Compensatory changes in CYP expression that occur in these models may also occur in polymorphic humans, or may complicate interpretation of ADME studies performed using these models. The loss of CAR causes significant changes in several CYPs probably due to loss of CAR-mediated constitutive regulation of these CYPs. Expression and activity changes include significant repression of Cyp2a and Cyp2b members with corresponding drops in 6α- and 16ß-testosterone hydroxylase activity. Further, the ratio of 6α-/15α-hydroxylase activity, a biomarker of sexual dimorphism in the liver, indicates masculinization of female CAR-null mice, suggesting a role for CAR in the regulation of sexually dimorphic liver CYP profiles. The loss of Cyp3a causes fewer changes than CAR. Nevertheless, there are compensatory changes including gender-specific increases in Cyp2a and Cyp2b. Cyp2a and Cyp2b were down-regulated in CAR-null mice, suggesting activation of CAR and potentially PXR following loss of the Cyp3a members. However, the loss of Cyp2b causes few changes in hepatic CYP transcript levels and almost no significant compensatory changes in protein expression or activity with the possible exception of 6α-hydroxylase activity. This lack of a compensatory response in the Cyp2b9/10/13-null mice is probably due to low CYP2B hepatic expression, especially in male mice. Overall, compensatory and regulatory CYP changes followed the order CAR-null > Cyp3a-null > Cyp2b-null mice.

Quantitative Polymerase Chain Reaction for Microbial Growth Kinetics of Mixed Culture System.

Microbial growth kinetics is often used to optimize environmental processes owing to its relation to the breakdown of substrate (contaminants). However, the quantification of bacterial populations in the environment is difficult owing to the challenges of monitoring a specific bacterial population within a diverse microbial community. Conventional methods are unable to detect and quantify the growth of individual strains separately in the mixed culture reactor. This work describes a novel quantitative PCR (qPCR)-based genomic approach to quantify each species in mixed culture and interpret its growth kinetics in the mixed system. Batch experiments were performed for both single and dual cultures of Pseudomonas putida and Escherichia coli K12 to obtain Monod kinetic parameters (µmax and Ks). The growth curves and kinetics obtained by conventional methods (i.e., dry weight measurement and absorbance reading) were compared with that obtained by qPCR assay. We anticipate that the adoption of this qPCR-based genomic assay can contribute significantly to traditional microbial kinetics, modeling practice, and the operation of bioreactors, where handling of complex mixed cultures is required.

Quantitative detection of single walled carbon nanotube in water using DNA and magnetic fluorescent spheres.

Carbon nanotubes (CNTs) possess unique properties that have led to an increase in their research and usage for a wide variety of fields. This growing demand of CNTs poses a major public health risk given its unregulated release into the environment. Unfortunately there is a significant information gap on the actual quantity of CNTs in the environment due to limitation of existing detection methods. This is mainly owing to the ubiquitous carbon chemistry of CNT. In response we developed a method (CNT-capture method) that is able to structurally differentiate CNT from other interference carbon materials in an aqueous medium. The affinity between single walled nanotubes (SWNTs) and specific single stranded DNA (ssDNA) was employed to capture SWNTs in water. SWNT-specific separation was obtained via magnetic separation. Dual fluorescent labels attached to sandwich ssDNA probes were used for quantification. The specific affinity between DNA and SWNTs was verified and no significant side-interactions were observed. With optimized incubation duration (30 min) and buffer composition (10(-7) % sodium dodecyl sulfate and pH 7.9), a calibration curve of quantification (R(2) = 0.90) was obtained with a range of SWNT concentration (0.05-10 µg/mL) against graphene as a planar analog. Comparison to other spectroscopy based methods was carried out to highlight the specificity and sensitivity of the presented method for CNT detection in aquatic sample.

Nonylphenol-mediated CYP induction is PXR-dependent: The use of humanized mice and human hepatocytes suggests that hPXR is less sensitive than mouse PXR to nonylphenol treatment.

Nonylphenol (NP), a by-product of alkylphenol ethoxylates, is a pervasive surfactant that activates the xenosensing nuclear receptor, the pregnane X-receptor (PXR) in transactivation assays in vitro. We are interested in determining if NP activates PXR in vivo, determining if hPXR and mPXR act similarly, and investigating the role of PXR in protecting individuals from NP. Wild-type (WT), PXR-null, and humanized PXR (hPXR) mice were treated with NP at 0, 50 or 75mg/kg/day for one week, and cytochrome P450 (CYP) induction, liver histopathology, and serum NP concentrations were examined. WT mice treated with NP showed induction of Cyp2b, and male-specific induction of Cyp2c and Cyp3a. CYPs were not induced in PXR-null mice, demonstrating that PXR is necessary for NP-mediated CYP induction. CAR-mediated CYP induction was not observed in the PXR-null mice despite previous data demonstrating that NP is also a CAR activator. hPXR mice only showed moderate Cyp induction, suggesting that hPXR is not as sensitive to NP as mPXR in vivo. NP-mediated Cyp3a induction from three human hepatocyte donors was not significant, confirming that hPXR is not very sensitive to NP-mediated CYP induction. Lastly, mice with PXR (mPXR and hPXR) showed lower NP serum concentrations than PXR-null mice treated with NP suggesting that PXR plays a role in decreasing liver toxicity by basally regulating phase I-III detoxification enzymes that promote the metabolism and elimination of NP. In summary, PXR is required for NP-mediated CYP-induction, mPXR mediates greater CYP induction than hPXR in vivo, and the presence of PXR, especially mPXR, is associated with altered histopathology and increased clearance of NP.

Constitutive androstane receptor -null mice are sensitive to the toxic effects of parathion: association with reduced cytochrome p450-mediated parathion metabolism [corrected].

Constitutive androstane receptor [corrected](CAR) is activated by several chemicals and in turn regulates multiple detoxification genes. Our research demonstrates that parathion is one of the most potent, environmentally relevant CAR activators with an EC(50) of 1.43 microM. Therefore, animal studies were conducted to determine whether CAR was activated by parathion in vivo. Surprisingly, CAR-null mice, but not wild-type (WT) mice, showed significant parathion-induced toxicity. However, parathion did not induce Cyp2b expression, suggesting that parathion is not a CAR activator in vivo, presumably because of its short half-life. CAR expression is also associated with the expression of several drug-metabolizing cytochromes P450 (P450). CAR-null mice demonstrate lower expression of Cyp2b9, Cyp2b10, Cyp2c29, and Cyp3a11 primarily, but not exclusively in males. Therefore, we incubated microsomes from untreated WT and CAR-null mice with parathion in the presence of esterase inhibitors to determine whether CAR-null mice show perturbed P450-mediated parathion metabolism compared with that in WT mice. The metabolism of parathion to paraoxon and p-nitrophenol (PNP) was reduced in CAR-null mice with male CAR-null mice showing reduced production of both paraoxon and PNP, and female CAR-null mice showing reduced production of only PNP. Overall, the data indicate that CAR-null mice metabolize parathion slower than WT mice. These results provide a potential mechanism for increased sensitivity of individuals with lower CAR activity such as newborns to parathion and potentially other chemicals due to decreased metabolic capacity.

Evaluation of respiratory symptoms and their possible association with residential indoor bioaerosol concentrations and other environmental influences.

The study discussed here evaluated the presence of self-reported respiratory symptoms and their association with indoor bioaerosol concentrations over a year-long study in the El Paso, Texas, region. The authors collected air samples from homes to assess seasonal differences in bacterial and fungal bioaerosol concentrations. They distributed a health questionnaire to the participating homeowner during each seasonal air sampling. The authors used this questionnaire to assess whether the homeowners were suffering from specific symptoms prior to each sampling. Descriptive statistics and logistic regressions were conducted to model the relationship among "high" reporters of symptoms, bioaerosols, and environmental factors. The authors collected evidence to support an association between indoor respirable bacterial concentrations and homeowners that reported at least eight respiratory symptoms (odds ratio [OR] = 1.10, p = .045). Smoking status, indoor humidity, and season also displayed associations with homeowners that reported at least eight respiratory symptoms (current smokers OR = 3.3, p = .042; indoor humidity OR = 1.5, p = .030; spring season OR = 7.2, p = .001; fall season OR = 3.4, p = .008).

Characterization of seasonal indoor and outdoor bioaerosols in the arid environment of El Paso, Texas.

The authors conducted a study in the El Paso, Texas, region to assess the seasonal bioaerosol concentrations in a convenience sample of one-story residences. The authors sampled the same houses for each season over the course of a year (March 2005 to February 2006) to determine bacterial and fungal concentrations. They used a two-stage ambient culturable sampler system to collect the bioaerosol samples. They took indoor and outdoor bioaerosol samples and studied meteorological conditions for each house at each season. The study found that most of the measured bioaerosol concentrations differed statistically by season (p < .05). The greatest concentrations throughout the year were found to occur in fine-sized indoor bacteria during the winter. Meteorological conditions were found not to significantly influence bioaerosol concentrations throughout the year.

Seasonal fine and coarse culturable fungal constituents and concentrations from indoor and outdoor air samples taken from an arid environment.

This study was undertaken to determine the normal indoor and outdoor airborne culturable fungal constituents and concentrations of an arid environment. Air samples were taken with two-stage, ambient, culturable sampler systems and analyzed for nine specific fungal genera from 50 homes as a repeated measure during each season of the year. These homes had no previous histories of indoor air quality issues. This study detected seasonal differences for the arid environment between different culturable fungal concentrations across the two size ranges. The highest concentrations were during fall, in the outdoor fine-size range. The lowest concentrations were the indoor coarse concentrations in the spring. From this study it can be concluded that Cladosporium spp. had the highest concentrations during fall in an arid environment. The overall findings suggest that Cladosporium had concentrations greater than the other genera evaluated, specifically, the fall outdoor fine concentrations. Seasonality was found to be a key factor in determining the variability of fungal constituents and concentrations within the arid indoor and outdoor environments. The fine-size range was 12 times and 6 times greater than the coarse-size range for indoor and outdoor samples, respectively, which accounted for the majority of fungal organisms. In addition, the results from this study in an arid climate differ from those conducted in a moister climate.

Isolation of Staphylococcus aureus and antibiotic-resistant Staphylococcus aureus from residential indoor bioaerosols.

OBJECTIVE: In this study we evaluated the levels of Staphylococcus aureus and antibiotic-resistant S. aureus in colony-forming units (CFU) per cubic meter of air. DESIGN: We used Andersen two-stage samplers to collect bioaerosol samples from 24 houses in El Paso, Texas, using tryptic soy agar as the collection media, followed by the replicate plate method on Chapman Stone selective medium to isolate S. aureus. The Kirby-Bauer disk diffusion method was used to determine antibiotic resistance to ampicillin, penicillin, and cefaclor, which represent two distinct classes of antibiotics. RESULTS: The average recovered concentration of respirable heterotrophic organisms found outside each home was 345.38 CFU/m3, with an average of 12.63 CFU/m3 for S. aureus. The average recovered concentration of respirable heterotrophic organisms found inside each home was 460.23 CFU/m3, with an average of 15.39 CFU/m3 for S. aureus. The respirable S. aureus recovered from inside each home had an average resistance of 54.59% to ampicillin and 60.46% to penicillin. Presence of cefaclor-resistant and of multidrug-resistant S. aureus was the same, averaging 13.20% per house. The respirable S. aureus recovered from outside each home had an average resistance of 34.42% to ampicillin and 41.81% to penicillin. Presence of cefaclor-resistant and of multidrug-resistant S. aureus was the same, averaging 13.96% per house. CONCLUSIONS: This study indicates that antibiotic-resistant bioaerosols are commonly found within residential homes. Our results also suggest that resistant strains of airborne culturable S. aureus are present in higher concentrations inside the study homes than outside the homes.

Isolation of antibiotic-resistant bacteria from the air plume downwind of a swine confined or concentrated animal feeding operation.

OBJECTIVE: In this study we evaluated the levels of antibiotic- and multidrug-resistant bacteria in bioaerosols upwind, within, and downwind at locations 25 m, 50 m, 100 m, and 150 m from a swine confined animal feeding operation. DESIGN: We used Andersen two-stage samplers to collect bacterial samples, the replicate plate method to isolate organisms, and the Kirby-Bauer disk diffusion method to determine antibiotic resistance. RESULTS: The percentage of organisms resistant to at least two antibiotic classes and all four classes evaluated were, respectively, 2.1 and 3.0 times higher inside (n = 69) than upwind (n = 59) of the facility. Staphylococcus aureus was the most prevalent organism recovered. Concentrations of antibiotic-resistant S. aureus decreased with increasing distance from the facility. Using Fisher's exact methods, the change in distribution of antibiotic resistance profiles for each antibiotic was statistically significant (oxytetracycline, p = 0.010; tetracycline, p = 0.014; ampicillin, p = 0.007; erythromycin, p = 0.035); however, this relationship was not seen with lincomycin and penicillin (p > 0.05) . In addition, the levels of antibiotic-resistant S.aureus 25 m downwind were significantly greater than the levels from samples taken upwind from the facility for the same four antibiotics (p < 0.05) . The percentage of resistant group A streptococci and fecal coliform increased within the facility compared with upwind values for all antibiotics evaluated,except for lincomycin. The percentage of resistant total coliform organisms increased within the facility compared with upwind values for oxytetracycline and tetracycline. CONCLUSIONS: Bacterial concentrations with multiple antibiotic resistances or multidrug resistance were recovered inside and outside to (at least) 150 m downwind of this facility at higher percentages than upwind. Bacterial concentrations with multiple antibiotic resistances were found within and downwind of the facility even after subtherapeutic antibiotics were discontinued. This could pose a potential human health effect for those who work within or live in close proximity to these facilities.

Bacterial plume emanating from the air surrounding swine confinement operations.

The objective of this study was to evaluate the levels of bacteria in the air plume immediately upwind at 25 m and downwind at locations 25 m, 50 m, 100 m, and 150 m from a confined animal feeding operation (CAFO). It was hypothesized that this would give insight into determining the maximal distance that bacterial organisms release from a CAFO could travel, which would be important in determining the optimal siting distance for future CAFO in relation to high population areas. The Andersen two-stage sampler was used to collect all of the bacterial samples from the animal confinement facilities. The data show a marked increase in bacterial CFUs/m3 inside the facility (18,132 CFU/m3 average) versus upwind (63 CFU/m3 average) anda steady down wind decrease out to approximately 150 m. Staphylococcus aureus was found to account for 76% of the organisms recovered. We conclude that the optimal placement of a swine CAFO would be at least 200 m from a residential area.