Formaldehyde is not only a widely used chemical with well-known carcinogenicity but is also a normal metabolite of living cells. It thus poses unique challenges for understanding risks associated with exposure. N 2-hydroxymethyl-dG (N 2 -HOMedG) is the main formaldehyde-induced DNA mono-adduct, which together with DNA-protein crosslinks (DPCs) and toxicityinduced cell proliferation, play important roles in a mutagenic mode of action for cancer. In this study, N 2 -HOMe-dG was shown to be an excellent biomarker for direct adduction of formaldehyde to DNA and the hydrolysis of DPCs. The use of inhaled [ 13 CD 2 ]-formaldehyde exposures of rats and primates coupled with ultrasensitive nano ultra performance liquid chromatography-tandem mass spectrometry permitted accurate determinations of endogenous and exogenous formaldehyde DNA damage. The results show that inhaled formaldehyde only reached rat and monkey noses, but not tissues distant to the site of initial contact. The amounts of exogenous adducts were remarkably lower than those of endogenous adducts in exposed nasal epithelium. Moreover, exogenous adducts accumulated in rat nasal epithelium over the 28-days exposure to reach steady-state concentrations, followed by elimination with a half-life (t 1/2 ) of 7.1 days. Additionally, we examined artifact formation during DNA preparation to ensure the accuracy of nonlabeled N 2 -HOMe-dG measurements. These novel findings provide critical new data for understanding major issues identified by the National Research Council Review of the 2010 Environmental Protection Agency's Draft Integrated Risk Information System Formaldehyde Risk Assessment. They support a data-driven need for reflection on whether risks have been overestimated for inhaled formaldehyde, whereas underappreciating endogenous formaldehyde as the primary source of exposure that results in bone marrow toxicity and leukemia in susceptible humans and rodents deficient in DNA repair.

DNA-protein crosslinks (DPCs) arise from a wide range of endogenous and exogenous chemicals, such as chemotherapeutic drugs and formaldehyde. Importantly, recent identification of aldehydes as endogenous genotoxins in Fanconi anemia has provided new insight into disease causation. Due to their bulky nature, DPCs pose severe threats to genome stability, but previous methods to measure formaldehyde-induced DPCs were incapable of discriminating between endogenous and exogenous sources of chemical. In this study, we developed methods that provide accurate and distinct measurements of both exogenous and endogenous DPCs in a structurally-specific manner. We exposed experimental animals to stable isotope-labeled formaldehyde ([13CD2]-formaldehyde) by inhalation and performed ultrasensitive mass spectrometry to measure endogenous (unlabeled) and exogenous (13CD2-labeled) DPCs. We found that exogenous DPCs readily accumulated in nasal respiratory tissues, but were absent in tissues distant to the site of contact. This observation together with the finding that endogenous formaldehyde-induced DPCs were present in all tissues examined suggests that endogenous DPCs may be responsible for increased risks of bone marrow toxicity and leukemia. Furthermore, the slow rate of DPC repair provided evidence for persistence of DPCs. In conclusion, our method for measuring endogenous and exogenous DPCs presents a new perspective for the potential health risks inflicted by endogenous formaldehyde, and may inform improved disease prevention and treatment strategies.

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Women are more resistant to hepatocellular carcinoma (HCC) than men despite equal exposure to major risk factors, such as hepatitis B or C virus infection. Female resistance is hormone-dependent, as evidenced by the sharp increase in HCC incidence in postmenopausal women who do not take hormone replacement therapy. In rodent models sex-dimorphic HCC phenotypes are pituitary-dependent, suggesting that sex hormones act via the gonadalhypophyseal axis. We found that the estrogen-responsive pituitary hormone prolactin (PRL), signaling through hepatocyte-predominant short-form prolactin receptors (PRLR-S), constrained TNF receptor-associated factor (TRAF)-dependent innate immune responses invoked by IL-1β, TNF-α, and LPS/Toll-like receptor 4 (TLR4), but not TRIF-dependent poly(I:C)/TLR3. PRL ubiquitinated and accelerated poststimulatory decay of a "trafasome" comprised of IRAK1, TRAF6, and MAP3K proteins, abrogating downstream activation of c-Myc-interacting pathways, including PI3K/AKT, mTORC1, p38 MAPK, and NF-κB. Consistent with this finding, we documented exaggerated male liver responses to immune stimuli in mice and humans. Tumor promotion through, but regulation above, the level of c-Myc was demonstrated by sex-independent HCC eruption in Alb-Myc transgenic mice. PRL deficiency accelerated liver carcinogenesis in Prl −/− mice of both sexes. Conversely, pharmacologic PRL mobilization using the dopamine D2 receptor antagonist domperidone prevented HCC in tumor-prone C3H/HeN males. Viewed together, our results demonstrate that PRL constrains tumor-promoting liver inflammation by inhibiting MAP3K-dependent activation of c-Myc at the level of the trafasome. PRL-targeted therapy may hold promise for reducing the burden of liver cancer in high-risk men and women.liver neoplasms | lactotrophs | innate immunity | sex dimorphism

The bacterial metabolite and transition metal chelator pyridine-2,6-dithiocarboxylic acid (PDTC), promotes a novel and effective means of dechlorination of the toxic and carcinogenic pollutant, carbon tetrachloride. Pyridine-2,6-dithiocarboxylic acid has been presumed to act as a siderophore in the Pseudomonas strains known to produce it. To explore further the physiological function of PDTC production, we have examined its regulation, the phenotype of PDTC-negative (pdt) mutants, and envelope proteins associated with PDTC in P. putida strain DSM 3601. Aspects of the regulation of PDTC production and outer membrane protein composition were consistent with siderophore function. Pyridine-2,6-dithiocarboxylic acid production was coordinated with production of the well-characterized siderophore pyoverdine; exogenously added pyoverdine led to decreased PDTC production, and added PDTC led to decreased pyoverdine production. Positive regulation of a chromosomal pdtI-xylE transcriptional fusion, and of a 66 kDa outer membrane protein (IROMP), was seen in response to exogenous PDTC. Tests with transition metal chelators indicated that PDTC could provide a benefit under conditions of metal limitation; the loss of PDTC biosynthetic capacity caused by a pdtI transposon insertion resulted in increased sensitivity to 1,10-phenanthroline, a chelator that has high affinity for a range of divalent transition metals (e.g. Fe(2+), Cu(2+), Zn(2+)). Exogenously added PDTC could also suppress a phenotype of pyoverdine-negative (Pvd-) mutants, that of sensitivity to EDDHA, a chelator with higher affinity and specificity for Fe(3+). Measurement of 59Fe incorporation showed uptake from 59Fe:PDTC by DSM 3601 grown in low-iron medium, but not by cells grown in high iron medium, or by the pdtI mutant, which did not show expression of the 66 kDa envelope protein. These data verified a siderophore function for PDTC, and have implicated it in the uptake of transition metals in addition to iron.

The presence of viable but nonculturable bacteria in human clean-catch and mouse bladder-isolated urine specimens was investigated. Viable but nonculturable bacteria are alive but do not give rise to visible growth under nonselective growth conditions. Urine specimens obtained from human female volunteers with or without an active urinary tract infection were found to contain, on average, significantly more viable than culturable forms of bacteria. Additional support for the presence of viable but nonculturable cells in urine specimens considered sterile was obtained from examination of urine specimens obtained directly from the bladder of healthy mice. Because the viability assay used to study the viable but nonculturable condition is by necessity growth independent, and hence indirect, the accuracy of this assay that scores cells with intact cell membranes as being viable was studied. Greater than 95% of Escherichia coli cells exposed to lethal doses of UV irradiation were found to lose their membrane integrity within a day, a time frame similar to that used to examine urine specimens. These data suggest that viable but nonculturable cells can occur within regions of the urinary tract previously considered sterile.Urinary tract infections (UTIs) affect as many as 50% of women at least once during their lifetime (29, 32), and 25% of those who acquire a UTI will have another infection within the following 6 months (17). A UTI occurs when the urinary tract is infected with microorganisms, and uropathogenic Escherichia coli accounts for greater than 80% of all UTI cases (4, 30). One method of diagnosing a UTI is by culturing urine specimens; a threshold of 100,000 CFU/ml in clean-catch urine specimens is considered to indicate a UTI (4, 28). This threshold is not an absolute indicator, as both asymptomatic bacteriuria and patients with UTI symptoms having no culturable urine bacteria occur (29,32).Urine within the urinary tract is generally considered sterile (14). This conclusion is based upon a lack of culturable cells present in urine specimens obtained via clean-catch and catheterization methods. The presence of viable bacteria in the urine specimens of healthy patients would impact on hypotheses to explain recurrent UTIs as well as diagnostic procedures. Most recurrent UTIs result from reinfection; however, a higher percentage than would be expected by chance are caused by the index strain (6,18,26,27,41). The physical location and physiological status of index strain cells that remain after successful antibiotic therapy are unknown. Observations with a mouse model indicate that uropathogenic E. coli cells can remain in the urinary tract following antibiotic ther-

Exposure to fine particulate matter (PM 2.5), of which secondary organic aerosol (SOA) is a major constituent, is linked to adverse health outcomes, including cardiovascular disease, lung cancer, and preterm birth. Atmospheric oxidation of isoprene, the most abundant nonmethane hydrocarbon emitted into Earth's atmosphere primarily from vegetation, contributes to SOA formation. Isoprene-derived SOA has previously been found to alter inflammatory/oxidative stress genes. MicroRNAs (miRNAs) are epigenetic regulators that serve as post-transcriptional modifiers and key mediators of gene expression. To assess whether isoprene-derived SOA alters miRNA expression, BEAS-2B lung cells were exposed to laboratory-generated isoprene-derived SOA constituents derived from the acid-driven multiphase chemistry of authentic methacrylic acid epoxide (MAE) or isomeric isoprene epoxydiols (IEPOX) with acidic sulfate aerosol particles. These IEPOX-and MAE-derived SOA constituents have been shown to be measured in large quantities within PM 25 collected from isoprene-rich areas affected by acidic sulfate aerosol particles derived from human activities. A total of 29 miRNAs were identified as differentially expressed when exposed to IEPOX-derived SOA and 2 when exposed to MAE-derived SOA, a

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