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Fixation and decalcification can alter protein structure in tissues, influencing the efficacy of primary antibodies routinely used in immunohistochemical (IHC) staining. Histologic examination of zebrafish requires both processes, making staining and analysis potentially challenging. Here, we investigated the effects of common fixation and decalcification protocols on IHC staining in zebrafish. We also identified zebrafish-reactive and -specific antibodies for use in research and diagnostics. For several of the antibodies, time spent in Dietrich's fixative containing 2% glacial acetic acid or 3.4% formaldehyde followed by decalcification with ethylenediaminetetraacetic acid (EDTA) significantly impacted IHC staining quality, particularly regarding staining intensity. Protocols utilizing shorter fixation times produced higher-quality stains. In addition, individual markers were variably affected by the type of fixative. Dietrich's fixative significantly reduced staining quality for the "neural" markers: glial fibrillar acidic protein, chromogranin A, S100. A negative time-dependent effect of fixation on staining quality was found for several antibodies: muscle actin (Dietrich's only), cytokeratin AE1/AE3, chromogranin, and S100. Neither decalcification protocol had a statistically significant negative time-dependent effect on staining quality. Based on our results, we suggest shorter fixation and decalcification protocols to best preserve IHC staining quality as well as recommend deliberate selection of the fixative used depending on the protein of interest.

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) targets multiple organs for tumorigenesis in the rat, including the colon and the skin. PhIP-induced skin tumors were subjected to mutation screening, which identified genetic changes in Hras (7/40, 17.5%) and Tp53 (2/40, 5%), but not in Ctnnb1, a commonly mutated gene in PhIP-induced colon tumors. Despite the absence of Ctnnb1 mutations, β-catenin was overexpressed in nuclear and plasma membrane fractions from PhIP-induced skin tumors, coinciding with loss of p120-catenin from the plasma membrane, and the appearance of multiple p120-catenin-associated bands in the nuclear extracts. Real-time RT-PCR revealed that p120-catenin isoforms 1 and 4 were upregulated in PhIP-induced skin tumors, whereas p120-catenin isoform 3 was expressed uniformly, compared with adjacent normal-looking tissue. In human epidermoid carcinoma and colon cancer cells, transient transfection of p120-catenin isoform 1A enhanced the viability and cell invasion index, whereas transient transfection of p120-catenin isoform 4A increased cell viability and cell proliferation. Knockdown of p120-catenin revealed a corresponding reduction in the expression of β-catenin and a transcriptionally regulated target, Ccnd1/Cyclin D1. Co-immunoprecipitation experiments identified associations of β-catenin with p120-catenin isoforms in PhIP-induced skin tumors and human cancer cell lines. The results are discussed in the context of therapeutic strategies that might target different p120-catenin isoforms, providing an avenue to circumvent constitutively active β-catenin arising via distinct mechanisms in skin and colon cancer.

Current assumption for assessing carcinogenic risk of polycyclic aromatic hydrocarbons (PAHs) is that they function through a common mechanism of action; however, recent studies demonstrate that PAHs can act through unique mechanisms potentially contributing to cancer outcomes in a non-additive manner. Using a primary human 3D bronchial epithelial culture (HBEC) model, we assessed potential differences in mechanism of toxicity for two PAHs, benzo[a]pyrene (BAP) and dibenzo[def,p]chrysene (DBC), compared to a complex PAH mixture based on short-term biosignatures identified from transcriptional profiling. Differentiated bronchial epithelial cells were treated with BAP (100–500 μg/ml), DBC (10 μg/ml), and coal tar extract (CTE 500–1500 μg/ml, SRM1597a) for 48 h and gene expression was measured by RNA sequencing or quantitative PCR. Comparison of BAP and DBC gene signatures showed that the majority of genes (~60%) were uniquely regulated by treatment, including signaling pathways for inflammation and DNA damage by DBC and processes for cell cycle, hypoxia and oxidative stress by BAP. Specifically, BAP upregulated targets of AhR, NRF2, and KLF4, while DBC downregulated these same targets, suggesting a chemical-specific pattern in transcriptional regulation involved in antioxidant response, potentially contributing to differences in PAH potency. Other processes were regulated in common by all PAH treatments, BAP, DBC and CTE, including downregulation of genes involved in cell adhesion and reduced functional measurements of barrier integrity. This work supports prior in vivo studies and demonstrates the utility of profiling short-term biosignatures in an organotypic 3D model to identify mechanisms linked to carcinogenic risk of PAHs in humans.

Eomesodermin (Eomes) is a T-box transcription factor that has been implicated in the etiology of colorectal cancer and other human malignancies. We screened a panel of human primary colon cancers and patient-matched controls (n = 30) and detected Eomes overexpression at the mRNA and protein level. Similar results were obtained in a panel of rat colon tumors and adjacent normal-looking colonic mucosa (n = 24). In human colon cancer cells, forced overexpression of Eomes enhanced cell viability and protected against staurosporine-induced apoptosis. On the other hand, knocking down Eomes resulted in reduced cell viability, G2/M cell cycle arrest, and apoptosis induction. The apoptotic mechanism centered on the reciprocal downregulation of anti-apoptotic BIRC5 (Survivin) and upregulation of proapoptotic Bcl-2 modifying factor (BMF). In patients with colorectal cancer, high EOMES expression (n = 95) was associated with poor overall survival compared with individuals exhibiting low EOMES levels (n = 80). We conclude from the current investigation, and prior literature, that Eomes has a divergent role in cancer development, with evidence for tumor suppressor and oncogenic functions, depending on stage and tissue context. Further studies are warranted on the apoptotic mechanisms linked to the reciprocal regulation of BMF and BIRC5 in human colorectal cancers characterized by Eomes overexpression.

Mycobacterium avium (MAC) lives and replicates in macrophages and causes disseminated disease in immunocompromised individuals. As a host response to control disease, many macrophages become apoptotic a few days after MAC infection. In this study, we hypothesized that MAC can survive autophagic and apoptotic macrophages and spread.Electron, time-lapse video, fluorescence microscopy. Apoptosis was determined by ELISA and TUNEL assays. Autophagy was seen by migration of LC3-1.Apoptotic macrophages harbor chiefly viable MAC. MAC escapes both the vacuole and the macrophage once apoptosis is triggered, leaving the bacteria free to infect nearby macrophages in the process of spreading. In addition, some MAC species will have apoptotic bodies and are released in healthy macrophages following apoptotic body ingestion. Because autophagy precedes apoptosis, it was established that heat-killed MAC, and viable MAC induces autophagy in macrophages at similar rates, but MAC still survives.MAC spreading from cell-to-cell is triggered by the macrophage's attempt to kill the bacterium, undergoing apoptosis.

NADPH oxidase/dual-oxidase (Nox/Duox) family members have been implicated in nuclear factor kappa-B (NFκB)-mediated inflammation and inflammation-associated pathologies. We sought to examine, for the first time, the role of Nox/Duox and NFκB in rats treated with the cooked meat heterocyclic amine carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). In the PhIP-induced colon tumors obtained after 1 year, Nox1, Nox4, NFκB-p50 and NFκB-p65 were all highly overexpressed compared with their levels in adjacent normal-looking colonic mucosa. Nox1 and Nox4 mRNA and protein levels also were markedly elevated in a panel of primary human colon cancers, compared with their matched controls. In HT29 human colon cancer cells, Nox1 knockdown induced G1 cell cycle arrest, whereas in Caco-2 cells there was a strong apoptotic response, with increased levels of cleaved caspase-3, -6, -7 and poly(ADP-ribose)polymerase. Nox1 knockdown blocked lipopolysaccharide-induced phosphorylation of IκB kinase, inhibited the nuclear translocation of NFκB (p50 and p65) proteins, and attenuated NFκB DNA binding activity. There was a corresponding reduction in the expression of downstream NFκB targets, such as MYC, CCND1 and IL1β. The results provide the first evidence for a role of Nox1, Nox4 and NFκB in PhIP-induced colon carcinogenesis, including during the early stages before tumor onset. Collectively, the findings from this investigation and others suggest that further work is warranted on the role of Nox/Duox family members and NFκB in colon cancer development.

Malignant round cell neoplasia was identified in 12 llamas and 12 alpacas aged 0–23 years. Mean age of affected alpacas (3.1 years) was significantly less than that of affected llamas (8.0 years). Tumor cell morphology varied from large and often pleomorphic (11 tumors) to small and often homogeneous (13 tumors). Neoplastic lesions were multicentric in 12 cases. Other sites were gastric (5 cases), intra-abdominal (perirenal; 4 cases), intrathoracic (2 cases), and cervical (1 case). Immunohistochemistry with antibodies to CD79α, BLA36, and CD3 identified B-cell lymphoma (12 cases) and T-cell lymphoma (6 cases). Six tumors did not express any lymphoid marker and were further immunostained for neuron-specific enolase (NSE), synaptophysin, S-100, glial fibrillary acidic protein (GFAP), and chromogranin A. All 6 of these tumors were negative for GFAP and chromogranin A but expressed 1 or more of the neural markers NSE, synaptophysin, and S-100 and were classified as primitive malignant round cell tumors (PMRCT). Tumor types could not be distinguished on the basis of animal age, gross pathologic appearance, tumor morphology, or tumor location. All animals with lymphoma and 5 with PMRCT died or were euthanatized. One alpaca with a focal cervical PMRCT lived for at least 20 months after diagnosis. Results of this study indicate that malignant round cell tumors in llamas and alpacas are a heterogeneous group that cannot be distinguished on the basis of signalment, postmortem findings, or routine light microscopic findings. Immunohistochemistry is a valuable diagnostic procedure when evaluating malignant round cell neoplasia in llamas and alpacas.

The carcinogenic potential of dibenzo[a,l]pyrene (DBP) has been well characterized in numerous animal models. We have previously documented that a single dose of 15 mg/Kg DBP to pregnant mice late in gestation (GD 17) produces an aggressive T-cell lymphoma as well as lung and liver cancer in offspring. The current study examines the chemopreventative properties of chlorophyllin (CHL) and chlorophyll (Chl) in this transplacental carcinogenesis model. Pregnant B6129SF1 females, bred to 129S1/SvIm males, received purified diets incorporated with either 2000 p.p.m. CHL, 2000 p.p.m. Chl or 10% freeze-dried spinach beginning at gestation day 9. Lymphoma-dependent mortality was not significantly altered by maternal consumption of any of the diet and little effect on lung tumor burden in mice surviving to 10 months of age was observed. However, coadministration of CHL at 380 mg/Kg with DBP by gavage (molar ratio of 10:1, CHL:DBP) provided significant protection against DBP-initiated carcinogenesis. Offspring born to dams receiving CHL co-gavaged with DBP exhibited markedly less lymphoma-dependent mortality (P < 0.001). The degree of protection by CHL, compared with controls dosed with DBP in tricaprylin (TCP) as the vehicle, was less marked, but still significant. Coadministration of CHL (TCP as vehicle) also reduced lung tumor multiplicity in mice by approximately 50% and this was observed throughout the study (P < 0.005). This is the first demonstration that CHL can provide potent chemoprotection in a transplacental carcinogenesis model and support a mechanism involving complex-mediated reduction of carcinogen uptake.

Dibenzo(a,l)pyrene (DBP) is among the most potent carcinogenic polycyclic aromatic hydrocarbons. Previously, we showed that DBP administration to pregnant mice resulted in high mortality of offspring from an aggressive T-cell lymphoma. All mice that survive to 10 months of age exhibit lung tumors with high multiplicity. Recombinant cytochrome P450 (cyp) 1b1 from mice and the homologue 1B1 in humans exhibit high activity toward the metabolic activation of DBP. Targeted disruption of the cyp1b1 gene protects against most DBP-dependent cancers. Mice heterozygous for the disrupted cyp1b1 allele were used to examine the effect of cyp1b1 gene dosage on DBP transplacental carcinogenesis. Dams were treated with 1 or 15 mg/kg of DBP or 50 mg/kg of benzo(a)pyrene. Cyp1b1-null offspring did not develop lymphoma, whereas wild-type and heterozygous siblings, born to dams given the high dose of DBP, exhibited significant mortalities between 10 and 30 weeks of age. At 10 months, all groups had lung adenomas or carcinomas [9.5%, 40.3%, 25.6%, and 100% incidences for controls, benzo(a)pyrene, 1 and 15 mg/kg DBP, respectively]. Cyp1b1 status did not alter benzo(a)pyrene-dependent carcinogenesis. At 1 mg/kg DBP, cyp1b1 status altered the incidence of lung tumors (19.0, 27.8, and 28.6% for nulls, heterozygous, and wild-type, respectively). At 15 mg/kg, tumor multiplicities in cyp1b1 wild-type (9.3) and heterozygous (9.5) offspring were nearly twice that of cyp1b1-null siblings (5.0). These data confirm that cyp1b1 bioactivation of DBP occurs in fetal target tissues, following transplacental exposure, with the thymus and lung as primary and secondary targets, respectively.

Both gemcitabine and synthetic double-stranded RNA (dsRNA) are known to be proapoptotic and immuno-stimulatory (-modulatory). We sought to evaluate the extent to which a combination therapy using gemcitabine and a synthetic dsRNA, polyinosine-cytosine (poly(I:C)), would improve the resultant anti-tumor activity. Using model lung and breast cancers in mice, we demonstrated that combination treatment of tumor-bearing mice with the poly(I:C) and gemcitabine synergistically delayed the tumor growth and prolonged the survival of the mice. The combination treatment also synergistically inhibited tumor cell growth in vitro and promoted more tumor cells to undergo apoptosis in vivo. Finally, the combination therapy generated a strong and durable specific anti-tumor immune response, although the immune response alone was unable to control the tumor growth after the termination of the therapy. This approach represents a promising therapy to improve the clinical outcomes for tumors sensitive to both dsRNA and gemcitabine.

Movement of glutamate receptors in neurons likely involves direct and indirect association of receptor subunits with microtubule- and actin-based motor proteins. We have previously shown that myosin II regulatory light chain (RLC) binds directly to subunits of the NMDA-type glutamate receptor (NR), suggesting that NMDA receptors are closely associated with a myosin II motor complex. Using a polyclonal antibody predicted to recognize all RLC isoforms previously described in rodent brain, we report the expression of RLC and the NR1 subunit in cortex, hippocampus and cerebellum of postnatal day 0 (P0) and adult mouse. Although myosin RLC was not exclusively localized with NR1 by immunohistochemistry, co-staining was striking in the neuronal soma of deep cortical neurons and Purkinje neurons of the cerebellum which showed a punctate, perinuclear pattern of immunoreactivity. These neuronal populations were identified using a monoclonal antibody directed against a nuclear-specific, transcriptional repressor, chicken ovalbumin upstream promoter-transcription factor (COUP-TF)-interacting protein 2 (CTIP2). Co-expression of NR1 and a myosin II motor was validated using an isoform specific anti-nonmuscle myosin II-B heavy chain (NMHC II-B) antibody. Our findings support the idea that there is regional heterogeneity in the molecular composition of the NMDA receptor-associated cytoskeleton, and suggest that NR subunits may be associated with an actin-based, myosin II-B motor within the endomembrane system of some neuronal populations. Differential staining patterns observed with light and heavy chain antibodies, however, suggest that there is also heterogeneity in the composition of myosin II complexes in brain.

An attractive approach to immunization is to apply DNA vaccine topically onto the skin. However, it is important to ensure that a strong immune response is induced without disrupting the skin stratum corneum. The hair follicles have been shown to be the major portal of entry for DNA applied onto the skin, and it has been reported that the transfection of hair follicle cells occurs mainly at the onset of a new growing stage of the hair cycle. Using an anthrax protective antigen (PA) protein-encoding plasmid in mice, we demonstrated that the anti-PA immune responses were significantly stronger when the hair follicles in the application area were induced into anagen-onset stage than when in telogen stage. The anti-PA antibodies enabled the immunized mice to survive a lethal dose of anthrax lethal toxin challenge. The enhanced immune responses can be partially attributed to the enhanced antigen gene expression and plasmid DNA uptake in the skin area wherein the hair follicles were induced into anagen-onset stage. Moreover, the moderate dermal inflammation associated with the anagen induction may also have contributed to the enhancement of the resultant immune response. This represents a novel approach to enhancing the immune response induced by a topically applied DNA vaccine.

There is growing interest in the possible health benefits of tea. We reported previously on the inhibition by white tea of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced colonic aberrant crypt foci (ACF) in the rat (4). To distinguish between blocking and suppressing effects, and thus provide mechanistic insights into prevention during the initiation versus post-initiation phases of carcinogenesis, white tea, and green tea were administered at 2% (w/v) as the sole source of drinking fluid either 2 wk before and 2 wk during PhIP dosing (100 mg/kg, every other day by oral gavage), or starting 1 wk after the carcinogen and continued until the study was terminated at 16 wk. In the former protocol, each tea produced marginal inhibition of colonic ACF, despite evidence for changes in several hepatic enzymes involved in heterocyclic amine metabolism. Post-initiation, however, the data were as follows (ACF/colon, mean +/- SE): PhIP/water 12.2 +/- 1.5; PhIP/white tea 5.9 +/- 0.9 (** P0.01); PhIP/caffeine 5.9 +/- 1.5 (** P0.01); PhIP/EGCG 3.5 +/- 0.8 (***P0.001); PhIP/green tea 8.9 +/- 1.2 (P = 0.22, not significant). In the latter study, apoptosis was determined using in situ oligo ligation and cleaved caspase-3 assays, whereas cell proliferation was assessed via bromodeoxyuridine (BrdU) incorporation. No consistent changes were seen in apoptosis assays, but BrdU labeling was as follows (percent of cells positive/colonic crypt, mean +/- SE): PhIP/water 10.4 +/- 0.6; PhIP/white tea 8.6 +/- 0.2 (*P0.05); PhIP/EGCG 6.0 +/- 0.85 (** P0.01); PhIP/caffeine 8.75 +/- 0.45 (*P0.05); PhIP/green tea 9.5 +/- 0.4 (P0.05, not significant). The data imply that white tea, caffeine, and EGCG may be most effective post-initiation, via the inhibition of cell proliferation in the colon and through the suppression of early lesions.

Serial sections of formalin-fixed, paraffin-embedded muscle biopsy specimens from 28 Quarter Horse, Paint, and draft-related breeds, aged 0.5-23 years, were treated with periodic acid-Schiff (PAS) stain for glycogen and were immunostained to detect ubiquitin expression. On the basis of findings in PAS-stained sections, a diagnosis of equine polysaccharide storage myopathy (EPSSM) was made in 22 horses aged 2-23 years (mean, 9.4 years); samples from 6 horses aged 0.5-15 years (mean, 7.3 years) had a normal PAS staining pattern, with no relevant lesions. Ubiquitin expression was detected in all but a 2-year-old EPSSM-affected horse and was not detected in the non-EPSSM-affected horses. Ubiquitin expression was greater than the degree of PAS-positive, amylase-resistant material, and ubiquitin was detected in aggregates of amylase-sensitive glycogen as well as in aggregates of amylase-resistant material. Results suggest that glycogen aggregates develop and are ubiquitinated prior to development of amylase-resistant inclusions. Ubiquitin immunostaining may be most useful for confirming the diagnosis of EPSSM in horses with only amylase-sensitive glycogen aggregates and in horses with early amylase-resistant inclusions. However, ubiquitin immunostaining is no more sensitive than is PAS staining for diagnosis of EPSSM.

Objective—To describe the microanatomic features of pancreatic islets and the immunohistochemical distribution of glucose transporter (GLUT) molecules in the pancreas and other tissues of New World camelids. Animals—7 healthy adult New World camelids, 2 neonatal camelids with developmental skeletal abnormalities, and 2 BALB/c mice. Procedure—Samples of pancreas, liver, skeletal muscle, mammary gland, brain, and adipose tissue were collected postmortem from camelids and mice. Pancreatic tissue sections from camelids were assessed microscopically. Sections of all tissues from camelids and mice (positive control specimens) were examined after staining with antibodies against GLUT-1, -2, -3, and -4 molecules. Results—In camelids, pancreatic islets were prominent and lacked connective tissue capsules. Numerous individual endocrine-type cells were visible distant from the islets. Findings in neonatal and adult tissues were similar; however, the former appeared to have more non–islet-associated endocrine cells. Via immunostaining, GLUT-2 molecules were detected on pancreatic endocrine cells and hepatocytes in camelids, GLUT-1 molecules were detected on the capillary endothelium of the CNS, GLUT-3 molecules were detected throughout the gray matter, and GLUT-4 molecules were not detected in any camelid tissues. Staining characteristics of neonatal and adult tissues were similar. Conclusions and Clinical Relevance—In New World camelids, microanatomic features of pancreatic islets are similar to those of other mammals. Data suggest that the poor glucose clearance and poor insulin response to hyperglycemia in adult camelids cannot be attributed to a lack of islet cells or lack of GLUT molecules on the outer membrane of those cells.

A 12-year-old intact female llama was euthanized following acute onset of spastic tetraparesis and recumbency with inability to rise. Postmortem examination revealed caudal cervical spinal cord compression due to a mass within the ventral spinal canal arising from the C6–C7 intervertebral disk space and attached to an irregularly thickened annulus fibrosis. On histopathologic examination, the mass was composed of amorphous acellular basophilic to amphophilic material admixed with irregularly arranged collagen bundles. The amorphous material was metachromatic and contained multiple small foci of markedly vacuolated round cells, characteristic of origin from the nucleus pulposus. Severe necrosis of all white matter tracts with astrocytic reaction was present in the overlying spinal cord segment. Ascending and descending Wallerian degeneration and dissecting interstitial astrogliosis were present within white matter tracts above and below the lesion, respectively. The diagnosis was compressive myelopathy due to chronic extrusion of the nucleus pulposus of the C6–C7 inter-vertebral disk. To the authors' knowledge, this is the first report of intervertebral disk disease in a camelid.

In mammals the cytochrome P450 3A (CYP3A) subfamily isoforms are primarily expressed in liver and intestines with lesser amounts found in other tissues. The aim of this study was to examine the Cellular localization and the expression pattern of CYP3A27 in the gastrointestinal tract (GI tract) of a freshwater teleost species, the rainbow trout (Oncorhynchus mykiss), A fish model used extensively for toxicological and carcinogenesis research. Using an avidin biotinylated enzyme complex and 3,3'-diaminobenzidine staining, strong cytoplasmic immunohistochemical staining was observed for CYP3A27 protein in hepatocytes and in enterocytes of the intestinal ceca and the proximal descending intestine when probed with a polyclonal antibody raised against rainbow trout P450 LMC5, a CYP3A protein. The intensity of epithelial staining decreased distally along the GI tract with faint staining observed in the epithelial cells examined near the vent. Western blot analysis was supportive of the immunohistochemistry results. Northern blot analysis also demonstrated that CYP3A27 mRNA was expressed along the entire GI tract. The major area of CYP3A27 mRNA expression was in the intestinal ceca, followed,by the proximal, descending intestine, at levels that were about three- to five-fold and two- to four-fold, respectively, greater than seen in the liver of the fish studied. Monooxygenase activities of intestinal ceca microsomes against testosterone and progesterone confirmed the presence of active CYP3A enzyme in this tissue. These results suggest that the intestine of rainbow trout may possesses substantial capacity for first-pass metabolism of xenobiotics by CYP3A27, which makes it an excellent model in which to study the consequence of such metabolism. (C) 2001 Elsevier Science.

Basic fibroblast growth factor (FGF) regulation of developmental markers in cell cultures derived from early zebrafish embryos was examined with the goal of in vitro culture of zebrafish embryonic stem cells and gaining an understanding of extracellular influences on early embryonic development. Markers were stem/primordial germ cell markers pou-2 and vas, neural markers zp-50, pax[zf-a], en-3, and wnt-1, and mesodermal markers gsc and myoD. Previously we had shown that FGF prevents the development of zebrafish pigment cells in vitro. In our culture system, FGF reduced expression of neural-specific markers, possibly implicating the FGF family in suppression of early neural cell development. Exposure to FGF for 24 hours at the time of seeding the cells was sufficient to suppress neural marker expression for a subsequent 4 days of culture, while absence of FGF for the first 24 hours of culture nullified the effect of FGF added subsequently. FGF predictably increased expression of gsc and myoD. Vas expression was unaffected, while pou-2 expression decreased with time in culture in the presence or absence of FGF. However, in situ hybridization identified a subpopulation of cells expressing pou-2, suggesting the possible continued existence of undifferentiated stem cells in the cultures.

γ-Glutamyltranspeptidase (GGT)-deficient mice (GGT−/−) display chronic glutathione (GSH) deficiency, growth retardation, and die at a young age (

During a 2-year period (1995-1997), vacuoles were detected in neurons of 21/50 (42% preva- lence) raccoons (Procyon lotor) in Oregon. Age or sex predisposition was not apparent. Twenty of these raccoons were from within a radius of 40 km of Corvallis in western Oregon. Microscopically, the vacuoles were variable in size, were in the perikarya, and were consistently present in pontine nuclei. Brain tissues were negative for rabies virus antigen by fluorescent antibody test and for the protease-resistant protein prion by immunohistochemistry. Electron microscopic examination of the brain stem of selected animals revealed ac- cumulation of electron-dense material within neuronal perikarya. Light and electron microscopic examination indicated that the accumulated intracellular material had a high lipid content. These lesions suggest a form of neuronal storage condition. Further research is required to identify the composition of the intracellular lipid material, to elucidate the mechanism of neuronal vacuolation in raccoons, and to understand the basis for the apparent geographic restriction of this lesion. Raccoons (Procyon lotor) are nocturnal and are widespread throughout North America. They are high- ly adaptable omnivores and have managed to survive and increase their numbers in many urban and subur- ban areas. For this reason, they have been used as monitors of zoonotic diseases and environmental con- tamination. 2,7 Spongiform changes affecting the neuropil and neu- ronal cell bodies have been described in experimental and natural cases of rabies in skunks and foxes 3 and in a heifer. 5 Neuronal perikaryonic vacuolation is also seen in scrapie and related spongiform encephalopa- thes 6 and in lysosomal and acquired storage diseases of various animals. 11 Naturally occurring transmissible spongiform encephalopathy and storage diseases have not been documented in raccoons, but rabies is com- monly encountered in raccoons of the eastern United States. However, vacuolation of neurons or the neu- ropil has not been seen in raccoons with naturally ac- quired rabies 9 or in raccoons experimentally infected with either a dog or a raccoon isolate of the rabies virus. 10 Recently vacuolation in neurons of 2 raccoons from 2 different geographic locations in the USA has been described. 8 Here, we describe histopathologic changes in 21 additional raccoons from the same geographic area of Oregon from which 1 of the original cases was identified. 9

The metazoan parasitic blood flukes, Schistosoma spp., infect over 200 million people worldwide and cause extensive human morbidity and mortality. Research strategies for development of anti-schistosomal agents are impeded by the organism’s complex molluscan–mammalian life cycle, which limits experimental approaches and availability of material. We derived long-term continuously proliferative cultures of Schistosoma mansoni sporocysts capable of generating cercariae in vitro . Cultured organisms retained the ability to parasitize the host, and they exhibited developmental regulation of candidate stage-specific genes in the host-free culture system. Evidence for expression of a reverse transcriptase also was found in the cultured organisms, pointing to this activity as a possible mechanistic contributor to the dynamic relationship between the parasite and its hosts. Continuous in vitro propagation of the asexual sporocyst stage allows isolation of clonally derived parasite populations and provides a means to study schistosomal molecular genetics, metabolism, and evasion of host defenses.

Endothelin-1 (ET-1) and its receptors are overexpressed in human cancers, but much less is known about the roles of ET-2 and ET-3 in cancer etiology. We sought to examine human and rat colon tumors for dysregulation of ET-2 and ET3 expression, and determine the underlying mechanisms. Human primary colon cancers and carcinogen-induced rat colon tumors were subjected to real-time RT-PCR, immunoblotting, and immunohistochemistry; EDN2 and EDN3 genes were examined by methylation-specific PCR, bisulfite sequencing, and pyrosequencing; and forced expression of ET-2 and ET-3 was conducted in human colon cancer cells followed by real-time cell migration and invasion assays. Rat and human colon tumors had markedly reduced expression of ET-2 and ET-3 mRNA and protein compared with matched controls. Mechanistic studies revealed hypermethylation of EDN2 and EDN3 genes in human primary colon cancers, and in a panel of human colon cancer cell lines. Forced expression of ET-2 and ET-3 attenuated significantly the migration and invasion of human colon cancer cells. We conclude that epigenetic inactivation of ET-2 and ET-3 occurs frequently in both rat and human colon cancers. Current therapeutic strategies target overexpressed members of the ET axis via small molecule inhibitors and receptor antagonists, but this work supports a complementary approach based on the re-expression of ET-2 and ET-3 as natural antagonists of ET-1 in colon cancer.

Inclusions of periodic acid-Schiff-positive, amylase resistant material were found within skeletal muscle fibers adjacent to an osteosarcoma in the proximal femur of an 8-year-old intact female Cocker Spaniel dog (dog No. 1) and adjacent to a synovial cell sarcoma of the stifle joint in a 7-year-old spayed female Bouvier des Flandres dog (dog No. 2). Inclusions were pale blue-gray with hematoxylin and eosin stain and formed irregular inclusions, replacing up to approximately 80% of the fiber diameter. Inclusions from dog No. 2 were of non-membrane-bound granular to filamentous material that occasionally formed discrete, elongate electrondense masses. The features of these inclusions were similar to those of materials previously described as complex polysaccharide, polyglucosan bodies, amylopectin, and Lafora bodies. Evidence for a generalized metabolic disorder was not found in these two dogs, suggesting that storage of complex polysaccharide can occur as a relatively nonspecific response to metabolic alterations in skeletal muscle in a variety of conditions.

Multiple, discrete, nodular foci of cartilaginous metaplasia were found in the spleens and kidneys of rockfishes taken from the northeastern Pacific Ocean during a survey to determine the incidence and the nature of diseases in these animals. These nodules sometimes occurred in association with granulomatous inflammation and distinct granulomas. Many of these fish were infected by Ichythophonus spp. or acid-fact bacteria (presumably Mycobacteria spp.). Some of the metaplastic foci contained encapsulated accumulations of eosinophilic vesicles and basophilic granular debris, described by other authors as “cysts of unknown etiology,” which have been observed at different sites in a variety of temperate and tropical fish species.

A novel swine-origin H1N1 influenza A virus has been identified as the cause of the 2009 influenza pandemic in humans. Since then, infections with the pandemic (H1N1) 2009 influenza virus have been documented in a number of animal species. The first known cases of lethal respiratory disease associated with pandemic (H1N1) 2009 influenza virus infection in house pets occurred in domestic cats in Oregon. A 10-year-old neutered domestic shorthair and an 8-year-old spayed domestic shorthair died shortly after developing severe respiratory disease. Grossly, lung lobes of both cats were diffusely firm and incompletely collapsed. Histologically, moderate to severe necrotizing to pyonecrotizing bronchointerstitial pneumonia was accompanied by serofibrinous exudation and hyaline membranes in the alveolar spaces. Influenza A virus was isolated from nasal secretions of the male cat and from lung homogenate of the female cat. Both isolates were confirmed as pandemic (H1N1) 2009 influenza virus by real-time reverse transcriptase polymerase chain reaction. With immunohistochemistry, influenza A viral antigen was demonstrated in bronchiolar epithelial cells, pneumocytes, and alveolar macrophages in pneumonic areas. The most likely sources of infection were people in the household with influenza-like illness or confirmed pandemic (H1N1) 2009 influenza. The 2 cases reported here provide, to the best of the authors’ knowledge, the first description of the pathology and viral antigen distribution of lethal respiratory disease in domestic cats after natural pandemic (H1N1) 2009 influenza virus infection, probably transmitted from humans.

The fetus and neonate cannot be viewed as 'little adults'; they are highly sensitive to toxicity from environmental chemicals. This phenomenon contributes to the fetal basis of adult disease. One example is transplacental carcinogenesis. Animal models demonstrate that environmental chemicals, to which pregnant women are daily exposed, can increase susceptibility of the offspring to cancer. It is uncertain to what degree in utero vs. lactational exposure contributes to cancer, especially for hydrophobic chemicals such as polyhalogenated biphenyls, ethers, dioxins, furans, etc., which can partition into breast milk. We developed a pregnant mouse model in which exposure to the polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]pyrene (DBP), during late gestation, produces an aggressive T-cell lymphoma in offspring between 3 and 6 months of age. Survivors exhibit multiple lung and liver (males) tumors. Here, we adopt a cross-foster design with litters born to dams treated with DBP exchanged with those born to dams treated with vehicle. Exposure to DBP in utero (about 2 days) produced significantly greater mortality than residual DBP exposure only through breast milk (3 weeks of lactation). As previously observed pups in all groups with an ahr{sup b-1/d} ('responsive') genotype were more susceptible to lymphoma mortality than ahr{sup d/d} ('non-responsive') siblings.more » At termination of the study at 10 months, mice exposed in utero also had greater lung tumor multiplicity than mice exposed only during lactation. Our results demonstrate that short exposure to DBP during late gestation presents a greater risk to offspring than exposure to this very hydrophobic PAH following 3 weeks of nursing.« less

Our laboratory recently developed a mouse model of transplacental induction of lymphoma, lung and liver cancer by the polycyclic aromatic hydrocarbon, dibenzo[a,l]pyrene (DBP). Pregnant B6129SF1 females, bred to 129S1/SvIm males, were treated on day 17 of gestation with an oral dose of 15 mg/kg DBP. Beginning on day 0 of gestation, dams were given (ad lib) buffered water, 0.5% green tea, 0.5% decaffeinated green tea, caffeine or epigallocatechin-3-gallate (EGCG) (both at equivalent concentrations found in tea). The concentration of the teas (and corresponding caffeine and EGCG) was increased to 1.0% upon entering the second trimester, 1.5% at onset of the third trimester and continued at 1.5% until pups were weaned at 21 days of age. Offspring were raised with normal drinking water and AIN93G diet. Beginning at 2 months of age, offspring experienced significant mortalities due to an aggressive T-cell lymphoma as seen in our previous studies. Ingestion of caffeinated, but not decaffeinated, green tea provided modest but significant protection (P = 0.03) against mortality. Caffeine provided a more robust (P = 0.006) protection, but EGCG was without effect. Offspring also developed DBP-dependent lung adenomas. All treatments significantly reduced lung tumor multiplicity relative to controls (P < 0.02). EGCG was most effective at decreasing tumor burden (P = 0.005) by on average over 40% compared with controls. Induction of Cytochrome P450 (Cyp)1b1 in maternal liver may reduce bioavailability of DBP to the fetus as a mechanism of chemoprevention. This is the first demonstration that maternal ingestion of green tea, during pregnancy and nursing, provides protection against transplacental carcinogenesis.

The carcinogenic effects of individual polycyclic aromatic hydrocarbons (PAH) are well established. However, their potency within an environmental complex mixture is uncertain. We evaluated the influence of diesel exhaust particulate matter on PAH-induced cytochrome P450 (CYP) activity, PAH-DNA adduct formation, expression of certain candidate genes and the frequency of tumor initiation in the two-stage Sencar mouse model. To this end, we monitored the effects of treatment of mice with diesel exhaust, benzo[a]pyrene (BP), dibenzo[a,l]pyrene (DBP), or a combination of diesel exhaust with either carcinogenic PAH. The applied diesel particulate matter (SRM(1975)) altered the tumor initiating potency of DBP: a statistically significant decrease in overall tumor and carcinoma burden was observed following 25 weeks of promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA), compared with DBP exposure alone. From those mice that were treated at the beginning of the observation period with 2 nmol DBP all survivors developed tumors (9 out of 9 animals, 100%). Among all tumors counted at the end, nine carcinomas were detected and an overall tumor incidence of 2.6 tumors per tumor-bearing animal (TBA) was determined. By contrast, co-treatment of DBP with 50mg SRM(1975) led to a tumor rate of only 66% (19 out of 29 animals), occurrence of only three carcinomas in 29 animals and an overall rate of 2.1 tumors per TBA (P=0.04). In contrast to the results with DBP, the tumor incidence induced by 200 nmol BP was found slightly increased when co-treatment with SRM(1975) occurred (71% vs. 85% after 25 weeks). Despite this difference in tumor incidence, the numbers of carcinomas and tumors per TBA did not differ statistically significant between both treatment groups possibly due to the small size of the BP treatment group. Since bioactivation of DBP, but not BP, predominantly depends on CYP1B1 enzyme activity, SRM(1975) affected PAH-induced carcinogenesis in an antagonistic manner when CYP1B1-mediated bioactivation was required. The explanation most likely lies in the much stronger inhibitory effects of certain PAHs present in diesel exhaust on CYP1B1 compared to CYP1A1. In the present study we also found molecular markers such as highly elevated AKR1C21 and TNFRSF21 gene expression levels in tumor tissue derived from animals co-treated with SRM(1975) plus DBP. Therefore we validate microarray data as a source to uncover transcriptional signatures that may provide insights into molecular pathways affected following exposure to environmental complex mixtures such as diesel exhaust particulates. V.M.A. and D.H.P. are members of ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility), a network of excellence operating with the European Union 6th Framework Program, Priority 5: Food Quality and Safety (Contract No. 513943).

Summary The cytoskeleton of cerebral microvascular endothelial cells is a critical determinant of blood-brain barrier (BBB) function. Barrier integrity appears to be particularly sensitive to the phosphorylation state of specific residues within myosin regulatory light chain (RLC), one of two accessory light chains of the myosin II motor complex. Phosphorylation of myosin RLC by myosin light chain kinase (MLCK) has been implicated in BBB dysfunction associated with alcohol abuse and hypoxia, whereas dephosphorylation may enhance BBB integrity following exposure to lipid-lowering statin drugs. Using immunohistochemistry we provide evidence of widespread myosin II RLC distribution throughout the cerebral vasculature of the mouse. Light microscopy revealed immunolocalization of myosin II RLC protein in the endothelium of brain capillaries, the endothelial cell layer of arterioles and in association with venules. Immunolabeling of myosin RLC in non-muscle endothelial cells could be distinguished from myosin RLC immunoreactivity associated with the smooth muscle layer of the tunica media in larger muscular arterioles. These findings support an emerging role for myosin II RLC as a component of the actomyosin cytoskeleton of cerebral endothelial cells with the potential to contribute to the selective vulnerability of the brain in vivo .

The polycyclic aromatic hydrocarbons (PAHs) benzo[a]pyrene (B[a]P) and dibenzo[a,l]pyrene (DB[a,l]P) are well-studied environmental carcinogens, however, their potency within a complex mixture is uncertain. We investigated the influence of urban dust particulate matter (UDPM) on the bioactivation and tumor initiation of B[a]P and DB[a,l]P in an initiation-promotion tumorigenesis model. SENCAR mice were treated topically with UDPM or in combination with B[a]P or DB[a,l]P, followed by weekly application of the promoter 12-O-tetradecanoylphorbol-13 acetate. UDPM exhibited weak tumor-initiating activity but significantly delayed the onset of B[a]P-induced tumor initiation by two-fold. When cotreated with UDPM, DB[a,l]P-treated animals displayed no significant difference in tumor-initiating activity, compared with DB[a,l]P alone. Tumor initiation correlated with PAH-DNA adducts, as detected by (33)P-postlabeling and reversed-phase high-performance liquid chromatography. Induction of cytochrome P450 (CYP)1A1 and 1B1 proteins was also detected following UDPM treatment or cotreatment with B[a]P or DB[a,l]P, indicating PAH bioactivation. Further genotoxicity analyses by the comet assay revealed that cotreatment of UDPM plus B[a]P or DB[a,l]P resulted in increased DNA strand breaks, compared with PAH treatment alone. The metabolizing activities of CYP1A1 and CYP1B1, as measured by the 7-ethoxyresorufin O-deethylation (EROD) assay, revealed that UDPM noncompetitively inhibited CYP1A1 and CYP1B1 EROD activity in a dose-dependent manner. Overall, these data suggest that components within complex mixtures can alter PAH-induced carcinogenesis by inhibiting CYP bioactivation and influence other genotoxic effects, such as oxidative DNA damage. These data further suggest that in addition to the levels of potent PAH, the effects of other mixture components must be considered when predicting human cancer risk.

Dietary modulation of cancer & cancer biomarkers. Dietary item or component studied: indole-3-carbinol (I3C)Outcome studied: lung tumor multiplicity. Study type: mice resulting from reciprocal crosses between B6129 SF1/J [AHR(b-1/d)] and 129Sv/ImJ [AHR(d/d)] mice. Tissue/biological material/sample size: heart, thymus, lung, spleen, liver, kidney, abnormal lymph node, testes or ovaries, colon and skinMode of exposure: dibenzo[a,l]pyrene (DBP) was administered to pregnant mice (15 mg/kg, gavage) on gestation day 17, and 2000 p.p.m. indole-3-carbinol (I3C) was fed to half of the mice from gestation day 9 until weaning. Impact on outcome (including dose-response): Offspring born to dams fed I3C exhibited markedly fewer mortalities (P < 0.0001). Maternal dietary exposure to I3C also significantly lowered lung tumor multiplicity (P = 0.035) in offspring surviving to 10 months of age. The I3C chemoprotection was independent of either maternal or fetal AHR genotype. The bioavailability of DBP to fetal target tissue was demonstrated by assessing DNA covalent adduction with a (33)P-post-labeling assay. The bioavailability of I3C was determined by dosing a subset of pregnant mice with [(14)C]-I3C. KEYWORD CLASSIFICATION: administration & dosage;analysis;Animals;antagonists & inhibitors;Anticarcinogenic Agents;Benzopyrenes;Biological Availability;chemically induced;Diet;dietary modulation of cancer & cancer biomarkers;DNA Adducts;Female;Fetal Diseases;humans;Indoles;Liver Neoplasms,Experimental;Lung;Lung Neoplasms;Lymphoma;Male;Maternal-Fetal Exchange;metabolism;Mice;mortality;NF-kappa B;nursing;pharmacokinetics;Pregnancy;prevention & control;Research;toxicity;Toxicology; The fetus and neonate are sensitive targets for chemically induced carcinogenesis. Few studies have examined the risk/benefit of chemoprotective phytochemicals, given in the maternal diet, against transplacental carcinogenesis. In this study, B6129 SF1/J (AHR(b-1/d)) and 129Sv/ImJ (AHR(d/d)) mice were cross-bred. The polycyclic aromatic hydrocarbon, dibenzo[a,l]pyrene (DBP), was administered to pregnant mice (15 mg/kg, gavage) on gestation day 17, and 2000 p.p.m. indole-3-carbinol (I3C), a chemoprotective phytochemical from cruciferous vegetables, was fed to half of the mice from gestation day 9 until weaning. Offspring born to dams fed I3C exhibited markedly fewer mortalities (P < 0.0001). Maternal dietary exposure to I3C also significantly lowered lung tumor multiplicity (P = 0.035) in offspring surviving to 10 months of age. The I3C chemoprotection was independent of either maternal or fetal AHR genotype. The bioavailability of DBP to fetal target tissue was demonstrated by assessing DNA covalent adduction with a (33)P-post-labeling assay. The bioavailability of I3C was determined by dosing a subset of pregnant mice with [(14)C]-I3C. Addition of chemoprotective agents to the maternal diet during pregnancy and nursing may be an effective new approach in reducing the incidence of cancers in children and young adults.

Lymphoma and leukemia are the most common cancers in children and young adults; in utero carcinogen exposure may contribute to the etiology of these cancers. A polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]pyrene (DBP), was given to pregnant mice (15 mg/kg body weight, gavage) on gestation day 17. Significant mortalities in offspring, beginning at 12 weeks of age, were observed due to an aggressive T-cell lymphoblastic lymphoma. Lymphocytes invaded numerous tissues. All mice surviving 10 months, exposed in utero to DBP, exhibited lung tumors; some mice also had liver tumors. To assess the role of the aryl hydrocarbon receptor (AHR) in DBP transplacental cancer, B6129SF1/J (AHRb-1/d, responsive) mice were crossed with strain 129S1/SvIm (AHRd/d, nonresponsive) to determine the effect of maternal and fetal AHR status on carcinogenesis. Offspring born to nonresponsive mothers had greater susceptibility to lymphoma, irrespective of offspring phenotype. However, when the mother was responsive, an AHR-responsive phenotype in offspring increased mortality by 2-fold. In DBP-induced lymphomas, no evidence was found for TP53, β-catenin, or Ki-ras mutations but lung adenomas of mice surviving to 10 months of age had mutations in Ki-ras codons 12 and 13. Lung adenomas exhibited a 50% decrease and a 35-fold increase in expression of Rb and p19/ARF mRNA, respectively. This is the first demonstration that transplacental exposure to an environmental PAH can induce a highly aggressive lymphoma in mice and raises the possibility that PAH exposures to pregnant women could contribute to similar cancers in children and young adults. (Cancer Res 2006; 66(2): 755-62)

Biomarkers of exposure & effect:: validationBiomarker: PhIPExposure/effect represented: PhIP/ G/C-T/A transversions Study type (in vitro, animals, humans): C57B1/6 miceMode of exposure (if in vivo) (acute, chronic, root of exposure): i.p. injectionsTissue/biological material/sample size: 4μm of tissuesDose-response: In Mlh1K/K mice, PhIP exposure increased the incidence of ACF from 31% (5/16) to 87% (13/15) (PZ0.003) and the average frequency of ACF from 0.4G0.2 to 2.7G0.6 (P! 0.0001)The PhIP-induced frequency of ACF in wild-type littermates was 0.6 per mouse (0.7G0.3-0.1G0.07; PZ0.04), associated with a corresponding increase in incidence from 7% (1/15) to 45% (5/11) (PZ0.05)The vehicle-treated Mlh1K/K mice also had an observed higher incidence (31 versus 7%; PZ0.17) and average frequency of ACF (0.4G0.2 versus 0.1G0.07; P! 0.08) than vehicle-treated. KEYWORDS CLASSIFICATION: Animals;chemically induced;Carcinogens;Carrier Proteins;Colon;Colonic Neoplasms;drug effects;dietary modulation of cancer & cancer biomarkers;DNA Mismatch Repair;DNA,Neoplasm;genetics;Hematologic Neoplasms;Imidazoles;Incidence;mechanisms of carcinogenesis;Mice;Mice,Knockout;Nuclear Proteins;pathology;physiology;Precancerous Conditions;Proteins;Research;Survival Rate;toxicity;Toxicology. Disruption of the DNA mismatch repair (MMR) pathway results in elevated mutation rates, inappropriate survival of cells bearing DNA damage, and increased cancer risk. Relatively little is known about the impact of environmentally relevant carcinogens on cancer risk in individuals with MMR-deficiency. We evaluated the effect of MMR status (Mlh1(+/+) versus Mlh1(-/-)) on the carcinogenic potential of the cooked-meat mutagen, 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) in mice. PhIP exposure did not obviously increase lymphoma or small intestinal tumorigenesis in either Mlh1-deficient or -proficient mice. In contrast, the frequency of aberrant crypt foci (ACF), a preneoplastic biomarker for colon tumorigenesis, was increased by PhIP, and the increase due to PhIP was significantly greater in Mlh1(-/-) versus wild-type littermates. This apparent heightened susceptibility to induction of ACF parallels the previously reported hypermutability of Mlh1-deficient mice to PhIP and is consistent with the hypothesis that MMR-deficiency would increase the likelihood of PhIP-induced carcinogenic mutations. Further evaluation of the risk that consumption of heterocyclic amines may impart to MMR-deficient individuals therefore is warranted.

Indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM) are naturally occurring dietary components found in cruciferous vegetables. In the stomach, I3C forms condensation products including DIM. I3C and DIM are marketed as dietary supplements, but little is known about the safety of long-term exposure. Rats were fed either control diet, 1 or 10x the current human dose of absorption-enhanced DIM or 5-7x the maximal recommended dose of I3C. Experimental diets were fed continuously for 3 or 12 months or 2 months followed by control diet for 1 month. Results at 3 or 12 months were similar in most respects. No significant differences between groups were found in blood chemistry. A general decrease in serum enzyme levels in male rats was observed, perhaps indicative of a protective effect. Males fed I3C exhibited higher serum levels of 25-hydroxy-vitamin D3 (25OH-D3). There were no observable differences grossly or histologically between groups, although a high number of hyaline casts were found throughout the kidneys of all animals. In both sexes total hepatic CYP levels were significantly induced by I3C, but not by either dose of DIM. Induction of CYP1A1 and CYP1A2 in liver and CYP1A1 in colon was detected for both sexes fed I3C and the high dose of DIM. CYP3A2 was induced in females fed I3C or the high dose of DIM; males were induced with I3C, but not DIM. No induction of CYP1B1 in the colon was observed in either sex. Long-term exposure to DIM produced no observable toxicity, and comparison to I3C indicates that DIM is a markedly less efficacious inducer of CYP in the rat at doses relevant to human supplementation.

Human exposure to polycyclic aromatic hydrocarbons (PAH) occurs through complex mixtures such as coal tar. The effect of complex PAH mixtures on the activation of carcinogenic PAH to DNA-binding derivatives and carcinogenesis were investigated in mice treated topically with NIST (National Institute of Standards and Technology) Standard Reference Material 1597 (SRM), a complex mixture of PAH extracted from coal tar, and either additional benzo[a]pyrene (B[a]P) or dibenzo[a,l]pyrene (DB[a,l]P). In an initiation-promotion study using 12-O-tetradecanoylphorbol-13-acetate as the promoter for 25 weeks, the SRM and B[a]P co-treated mice had a similar incidence of papillomas per mouse compared with the group exposed to B[a]P alone as the initiator. PAH-DNA adduct analysis of epidermal DNA by 33P-post-labeling and reversed-phase high-performance liquid chromatography found the SRM co-treatment led to a significant decrease in the total level of DNA adducts and B[a]P-DNA adducts to less than that observed in mice treated with B[a]P alone at 6, 12 and 72 h exposure. After 24 and 48 h exposure, there was no significant difference in the levels of adducts between these groups. In the DB[a,l]P initiation-promotion study, the co-treated group had significantly fewer papillomas per mouse than mice treated with DB[a,l]P alone as initiator. Averaging over the times of exposure gave strong evidence that mice co-treated with SRM and DB[a,l]P had a significantly lower level of PAH-DNA adducts than mice treated with DB[a,l]P alone. Western immunoblots showed that both cytochrome P450 (CYP) 1A1 and 1B1 were induced by the SRM. These results are consistent with the hypothesis that two major factors determining the carcinogenic activity of PAH within a complex mixture are (i) the persistence of certain PAH-DNA adducts as well as total adduct levels, and (ii) the ability of the components present in the mixture to inhibit the activation of carcinogenic PAH by the induced CYP enzymes.