Kay A. Fischer

In utero Exposure of Mice to Dibenzo(a,l )Pyrene Produces Lymphoma in the Offspring: Role of the Aryl Hydrocarbon Receptor

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 (AHR(b-1/d), responsive) mice were crossed with strain 129S1/SvIm (AHR(d/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, beta-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.

NADPH oxidase overexpression in human colon cancers and rat colon tumors induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)

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.

The influence of diesel exhaust on polycyclic aromatic hydrocarbon-induced DNA damage, gene expression and tumor initiation in Sencar mice in vivo

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.

Pathology and Viral Antigen Distribution of Lethal Pneumonia in Domestic Cats Due to Pandemic (H1N1) 2009 Influenza A Virus

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.

Chemoprevention of dibenzo[a,l]pyrene transplacental carcinogenesis in mice born to mothers administered green tea: primary role of caffeine

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.

Fetal Mouse Cyp1b1 and Transplacental Carcinogenesis from Maternal Exposure to Dibenzo(a,l)pyrene

Abstract 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.

Mycobacterium avium uses apoptotic macrophages as tools for spreading

BACKGROUND 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. METHODS Electron, time-lapse video, fluorescence microscopy. Apoptosis was determined by ELISA and TUNEL assays. Autophagy was seen by migration of LC3-1. RESULTS 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. CONCLUSION MAC spreading from cell-to-cell is triggered by the macrophages attempt to kill the bacterium, undergoing apoptosis.

Comparison of White Tea, Green Tea, Epigallocatechin-3-Gallate, and Caffeine as Inhibitors of PhIP-Induced Colonic Aberrant Crypts

Abstract: 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 (** P < 0.01); PhIP/caffeine 5.9 ± 1.5 (** P < 0.01); PhIP/EGCG 3.5 ± 0.8 (***P < 0.001); 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 (*P < 0.05); PhIP/EGCG 6.0 ± 0.85 (** P < 0.01); PhIP/caffeine 8.75 ± 0.45 (*P < 0.05); PhIP/green tea 9.5 ± 0.4 (P > 0.05, 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.

Epigenetic inactivation of endothelin‐2 and endothelin‐3 in colon cancer

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 ET‐3 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.

Malignant round cell neoplasia in llamas and alpacas.

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.