Combination Nivolumab, CD137 Agonism, and Adoptive Cell Therapy with Tumor-Infiltrating Lymphocytes for Patients with Metastatic Melanoma.

PURPOSE: Metastatic melanoma is a tumor amenable to immunotherapy in part due to the presence of antigen-specific tumor-infiltrating lymphocytes (TIL). These T cells can be activated and expanded for adoptive cell transfer (ACT), which has resulted in relatively high rates of clinical responses. Similarly, immune checkpoint inhibitors, specifically programmed cell death protein 1 (PD-1) blocking antibodies, augment antitumor immunity and increase the influx of T cells into tumors. Thus, we hypothesized that addition of PD-1 inhibition may improve the outcomes for patients undergoing ACT with TILs. PATIENTS AND METHODS: Patients with stage III/IV metastatic melanoma with unresectable disease who were anti-PD-1 treatment-naïve were enrolled. TILs were generated in the presence of anti-4-1BB antibody in vitro and expanded for ACT. Patients in cohort 1 received TIL infusion followed by nivolumab. Patients in cohort 2 also received nivolumab prior to surgical harvest and during TIL production. RESULTS: A total of 11 patients were enrolled, all of whom were evaluated for response, and nine completed ACT. Predominantly CD8+ TILs were successfully expanded from all ACT-treated patients and were tumor reactive in vitro. The trial met its safety endpoint, as there were no protocol-defined dose-limiting toxicity events. The objective response rate was 36%, and median progression-free survival was 5 months. Two nonresponders who developed new metastatic lesions were analyzed to determine potential mechanisms of therapeutic resistance, which included clonal divergence and intrinsic TIL dysfunction. CONCLUSIONS: Combination therapy with TILs and nivolumab was safe and feasible for patients with metastatic melanoma and provides important insights for future therapeutic developments in ACT with TILs.

Intratumoral delivery of dendritic cells plus anti-HER2 therapy triggers both robust systemic antitumor immunity and complete regression in HER2 mammary carcinoma.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) targeted antibodies in combination with chemotherapy has improved outcomes of HER2 positive (pos) breast cancer (BC) but toxicity of therapy remains a problem. High levels of tumor-infiltrating lymphocytes are associated with increased pathologic complete responses for patients treated with neoadjuvant therapy. Here we sought to investigate whether delivery of intratumoral (i.t.) multiepitope major histocompatibility complex (MHC) class II HER2 peptides-pulsed type I polarized dendritic cells (HER2-DC1) in combination with anti-HER2 antibodies without chemotherapy could enhance tumor regression by increasing anti-HER2 lymphocyte infiltration into the tumor. METHODS: BALB/c mice bearing orthotopic TUBO tumors, BALB/c mice bearing subcutaneous (s.c.) CT26 hHER2 tumors, or BALB-HER2/neu transgenic mice were all treated with i.t. or s.c. HER2-DC1, anti-HER2 antibodies, paclitaxel, T-DM1 or in combination. Immune response, host immune cells and effector function were analyzed using flow cytometry, interferon-γ ELISA and cytokine/chemokine arrays. The contributions of CD4+ and CD8+ T cells and antibody dependent cellular cytotoxicity (ADCC) were assessed using depleting antibodies and FcγR KO mice. Molecular changes were evaluated by immunohistochemistry and western blot. RESULTS: HER2-DC1 combined with anti-HER2 antibodies delivered i.t. compared to s.c. induced complete tumor regression in 75-80% of treated mice, with increased tumor infiltrating CD4+ and CD8+ T, B, natural killer T cells (NKT) and natural killer cells, and strong anti-HER2 responses in all HER2pos BC models tested. The therapy caused regression of untreated distant tumors. Labeled HER2-DC1 migrated prominently into the distant tumor and induced infiltration of various DC subsets into tumors. HER2-DC1 i.t. combined with anti-HER2 antibodies displayed superior antitumor response compared to standard chemotherapy with anti-HER2 antibodies. Lasting immunity was attained which prevented secondary tumor formation. The presence of CD4+ and CD8+ T cells and ADCC were required for complete tumor regression. In the HER2pos BC models, HER2-DC1 i.t. combined with anti-HER2 antibodies effectively diminished activation of HER2-mediated oncogenic signaling pathways. CONCLUSIONS: HER2-DC1 i.t. with anti-HER2 antibodies mediates tumor regression through combined activation of T and B cell compartments and provides evidence that HER2-DC1 i.t. in combination with anti-HER2 antibodies can be tested as an effective alternative therapeutic strategy to current chemotherapy and anti-HER2 antibodies in HER2pos BC.

Disseminated cancer cells in breast cancer: Mechanism of dissemination and dormancy and emerging insights on therapeutic opportunities.

Metastatic spread in breast cancer patients is the major driver of cancer-related deaths. A unique subset of cells disseminated from pre-invasive or primary tumor lesions are recognized as the main seeds for metastatic outgrowth. Disseminated cancer cells (DCCs) can migrate to distant organs and settle in a dormant state for a prolonged period until they emerge to overt metastases. Understanding the biology of breast cancer cells dissemination, dormancy and reactivation to form overt metastases has become an important focus. In this review, we discuss the recent advancements of molecular pathways involving breast cancer cell dissemination, role of chemokine-chemokine receptor networks in DCCs migration, DCCs phenotypic heterogeneity and unique genes signatures in tumor dormancy, microenvironmental regulation and specific niches that favors DCCs homing and dormancy. In addition, we also discuss recent findings relating to the role of immune response on DCC dissemination and dormancy. With recent advances in the field of immunotherapy/targeted therapy and its beneficial effects in cancer treatment, this review will focus on their impact on DCCs, reversal of stemness, tumor dormancy and metastatic relapse.

Th1 cytokine interferon gamma improves response in HER2 breast cancer by modulating the ubiquitin proteasomal pathway.

HER2 breast cancer (BC) remains a significant problem in patients with locally advanced or metastatic BC. We investigated the relationship between T helper 1 (Th1) immune response and the proteasomal degradation pathway (PDP), in HER2-sensitive and -resistant cells. HER2 overexpression is partially maintained because E3 ubiquitin ligase Cullin5 (CUL5), which degrades HER2, is frequently mutated or underexpressed, while the client-protective co-chaperones cell division cycle 37 (Cdc37) and heat shock protein 90 (Hsp90) are increased translating to diminished survival. The Th1 cytokine interferon (IFN)-γ caused increased CUL5 expression and marked dissociation of both Cdc37 and Hsp90 from HER2, causing significant surface loss of HER2, diminished growth, and induction of tumor senescence. In HER2-resistant mammary carcinoma, either IFN-γ or Th1-polarizing anti-HER2 vaccination, when administered with anti-HER2 antibodies, demonstrated increased intratumor CUL5 expression, decreased surface HER2, and tumor senescence with significant therapeutic activity. IFN-γ synergized with multiple HER2-targeted agents to decrease surface HER2 expression, resulting in decreased tumor growth. These data suggest a novel function of IFN-γ that regulates HER2 through the PDP pathway and provides an opportunity to impact HER2 responses through anti-tumor immunity.

Reactive Myelopoiesis Triggered by Lymphodepleting Chemotherapy Limits the Efficacy of Adoptive T Cell Therapy.

Adoptive T cell therapy (ACT) in combination with lymphodepleting chemotherapy is an effective strategy to induce the eradication of tumors, providing long-term regression in cancer patients. Despite that lymphodepleting regimens condition the host for optimal engraftment and expansion of adoptively transferred T cells, lymphodepletion concomitantly promotes immunosuppression during the course of endogenous immune recovery. In this study, we have identified that lymphodepleting chemotherapy initiates the mobilization of hematopoietic progenitor cells that differentiate to immunosuppressive myeloid cells, leading to a dramatic increase of peripheral myeloid-derived suppressor cells (MDSCs). In melanoma and lung cancer patients, MDSCs rapidly expanded in the periphery within 1 week after completion of a lymphodepleting regimen and infusion of autologous tumor-infiltrating lymphocytes (TILs). This expansion was associated with disease progression, poor survival, and reduced TIL persistence in melanoma patients. We demonstrated that the interleukin 6 (IL-6)-driven differentiation of mobilized hematopoietic progenitor cells promoted the survival and immunosuppressive capacity of post-lymphodepletion MDSCs. Furthermore, the genetic abrogation or therapeutic inhibition of IL-6 in mouse models enhanced host survival and reduced tumor growth in mice that received ACT. Thus, the expansion of MDSCs in response to lymphodepleting chemotherapy may contribute to ACT failure, and targeting myeloid-mediated immunosuppression may support anti-tumor immune responses.

Sequential Anti-PD1 Therapy Following Dendritic Cell Vaccination Improves Survival in a HER2 Mammary Carcinoma Model and Identifies a Critical Role for CD4 T Cells in Mediating the Response.

Patients with metastatic HER2 breast cancer (MBC) often become resistant to HER 2 targeted therapy and have recurrence of disease. The Panacea trial suggested that HER2 MBC patients were more likely to respond to checkpoint therapy if TIL were present or if tumor expressed PD-L1. We assessed whether type I polarized dendritic cells (DC1) could improve checkpoint therapy in a preclinical model of HER2+ breast cancer. TUBO bearing mice were vaccinated with either MHC class I or class II HER2 peptide pulsed DC1 (class I or class II HER2-DC1) concurrently or sequentially with administration of anti-PD-1 or anti-PDL1. Infiltration of tumors by immune cells, induction of anti-HER2 immunity and response to therapy was evaluated. Class I or class II HER2-DC1 vaccinated mice generated anti-HER2 CD8 or CD4+ T cell immune responses and demonstrated delayed tumor growth. Combining both MHC class I and II HER2-pulsed DC1 did not further result in inhibition of tumor growth or enhanced survival compared to individual administration. Interestingly class II HER2-DC1 led to both increased CD4 and CD8 T cells in the tumor microenvironment while class I peptides typically resulted in only increased CD8 T cells. Anti-PD-1 but not anti-PD-L1 administered sequentially with class I or class II HER2-DC1 vaccine could improve the efficacy of HER2-DC1 vaccine as measured by tumor growth, survival, infiltration of tumors by T cells and increase in systemic anti-HER2 immune responses. Depletion of CD4+ T cells abrogated the anti-tumor efficacy of combination therapy with class II HER2-DC1 and anti-PD-1, suggesting that tumor regression was CD4 dependent. Since class II HER2-DC1 was as effective as class I, we combined class II HER2-DC1 vaccine with anti-rat neu antibodies and anti-PD-1 therapy. Combination therapy demonstrated further delay in tumor growth, and enhanced survival compared to control mice. In summary, Class II HER2-DC1 drives both a CD4 and CD8 T cell tumor infiltration that leads to increased survival, and in combination with anti-HER2 therapy and checkpoint blockade can improve survival in preclinical models of HER2 positive breast cancer and warrants exploration in patients with HER2 MBC.

Immunotherapy in breast cancer: Current status and future directions.

Breast cancer, one of the leading causes of death in women in the United States, challenges therapeutic success in patients due to tumor heterogeneity, treatment resistance, metastasis and disease recurrence. Knowledge of immune system involvement in normal breast development and breast cancer has led to extensive research into the immune landscape of breast cancer and multiple immunotherapy clinical trials in breast cancer patients. However, poor immunogenicity and T-cell infiltration along with heightened immunosuppression in the tumor microenvironment have been identified as potential challenges to the success of immunotherapy in breast cancer. Oncodrivers, owing to their enhanced expression and stimulation of tumor cell proliferation and survival, present an excellent choice for targeted immunotherapy development in breast cancer. Loss of anti-tumor immune response specific to oncodrivers has been reported in breast cancer patients as well. Dendritic cell vaccines have been tested for their efficacy in generating anti-tumor T-cell response against specific tumor-associated antigens and oncodrivers and have shown improved survival outcome in patients. Here, we review the current status of immunotherapy in breast cancer, focusing on dendritic cell vaccines and their therapeutic application in breast cancer. We further discuss future directions of breast cancer immunotherapy and potential combination strategies involving dendritic cell vaccines and existing chemotherapeutics for improved efficacy and better survival outcome in breast cancer.

Oncodriver inhibition and CD4+ Th1 cytokines cooperate through Stat1 activation to induce tumor senescence and apoptosis in HER2+ and triple negative breast cancer: implications for combining immune and targeted therapies.

In patients with HER2-expressing breast cancer many develop resistance to HER2 targeted therapies. We show that high and intermediate HER2-expressing cancer cell lines are driven toward apoptosis and tumor senescence when treated with either CD4+ Th1 cells, or Th1 cytokines TNF-α and IFN-γ, in a dose dependent manner. Depletion of HER2 activity by either siRNA or trastuzumab and pertuzumab, and subsequent treatment with either anti-HER2 Th1 cells or TNF-α and IFN-γ resulted in synergistic increased tumor senescence and apoptosis in cells both sensitive and cells resistant to trastuzumab which was inhibited by neutralizing anti-TNF-α and IFN-γ. Th1 cytokines induced minimal senescence or apoptosis in triple negative breast cancer cells (TNBC); however, inhibition of EGFR in combination with Th1 cytokines sensitized those cells causing both senescence and apoptosis. TNF-α and IFN-γ led to increased Stat1 phosphorylation through serine and tyrosine sites and a compensatory reduction in Stat3 activation. Single agent IFN-γ enhanced Stat1 phosphorylation on tyrosine 701 and similar effects were observed in combination with TNF-α and EGFR inhibition. These results demonstrate Th1 cytokines and anti-oncodriver blockade cooperate in causing tumor senescence and apoptosis in TNBC and HER2-expressing breast cancer, suggesting these combinations could be explored as non-cross-reactive therapy preventing recurrence in breast cancer.

Mitochondrial ß-Carotene 9',10' Oxygenase Modulates Prostate Cancer Growth via NF-κB Inhibition: A Lycopene-Independent Function.

Despite numerous inquiries into protective roles of lycopene in prostate cancer prevention or therapy, little is known about mechanisms by which lycopene or its metabolites inhibit prostate cancer. The enzyme ß-carotene 9',10'-oxygenase (BCO2), which catalyzes asymmetric cleavage of several carotenoids, is the principal regulator of lycopene metabolism, but the range of BCO2 biological functions is incompletely understood. This study investigated expression and functional roles of BCO2 in human prostate cancer. Expression of the bco2 gene is dramatically decreased in prostate cancer tissue and in a range of prostate cancer cell lines as compared with nonneoplastic prostate tissue and normal prostatic epithelial cells, respectively. Inhibition of DNA methyltransferase activity restored bco2 expression in prostate cancer cell lines tested. Treatment with lycopene or its metabolite, apo-10-lycopenal, also increased bco2 expression and reduced cell proliferation in androgen-sensitive cell lines, but lycopene neither altered bco2 expression nor cell growth in androgen-resistant cells. Notably, restoring bco2 expression in prostate cancer cells inhibited cell proliferation and colony formation, irrespective of lycopene exposure. Exogenous expression of either wild-type BCO2 or a mutant (enzymatically inactive) BCO2 in prostate cancer cells reduced NF-κB activity and decreased NF-κB nuclear translocation and DNA binding. Together, these results indicate epigenetic loss of BCO2 expression is associated with prostate cancer progression. Moreover, these findings describe previously unanticipated functions of BCO2 that are independent of its enzymatic role in lycopene metabolism. IMPLICATIONS: This study identifies BCO2 as a tumor suppressor in prostate cancer. BCO2-mediated inhibition of NF-κB signaling implies BCO2 status is important in prostate cancer progression. Mol Cancer Res; 14(10); 966-75. ©2016 AACR.

The effects of maternal dietary docosahexaenoic acid intake on rat pup myelin and the auditory startle response.

UNLABELLED: We investigated the effects of maternal docosahexanoic acid (DHA) supplementation on pups' auditory startle responses and the composition of brain myelin. METHODS: Timed-pregnant rats were fed throughout pregnancy and lactation diets that contained 0, 0.3, 0.7 or 3% of total fatty acids as DHA. Milk was collected from culled pups' stomachs on postnatal day (PND) 3, latency of the auditory startle reflex was measured on PND 15, and pups were killed and brains collected on PND 24. RESULTS: Higher levels of DHA in maternal diet were reflected in milk and in pups' myelin. The latency of the auditory startle response was significantly longer in offspring of dams fed higher levels of DHA. There was a positive correlation between the myelin content of DHA and the latency of the startle response (p = 0.044), and a negative correlation between the myelin content of DHA and the myelin content of cholesterol (p = 0.005). CONCLUSION: High levels of maternal DHA intake alter the lipid composition of rat pup myelin, and are associated with longer latencies of the auditory startle response--a myelin-dependent electrophysiologic response.

Genotype-phenotype correlation between the polymorphic UGT2B17 gene deletion and NNAL glucuronidation activities in human liver microsomes.

The nicotine-derived tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is one of the most potent and abundant procarcinogens found in tobacco and tobacco smoke, and glucuronidation of its major metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), is an important mechanism for NNK detoxification. In cigarette smokers and tobacco chewers, there is a wide variation in the urinary levels of the ratio of NNAL to NNAL glucuronide (NNAL-Gluc). To determine whether genetic variation plays a potential role in this inter-individual variability, NNAL-glucuronidating activities were analysed in a series of human liver microsomal specimens and compared with UGT2B17 deletion genotypes in the same subjects. Assays performed in vitro demonstrated that over-expressed UGT2B17 exhibits high O-glucuronidating activity against NNAL. When stratifying subjects by UGT2B17 genotype, a significant or near-significant decrease in NNAL-O-Gluc formation was observed in liver microsomes from individuals who were either heterozygous [(+/0), P=0.07] or homozygous [(0/0), P=0.016] for the UGT2B17 deletion compared to liver microsomes from individuals with intact UGT2B17 alleles [(+/+)]. There was a significant (P<0.01) association between the level of liver microsomal NNAL-O-glucuronide formation and increasing numbers of the UGT2B17 null alleles in the liver microsomal specimens examined in this study, and a significant decrease in NNAL-O-Gluc formation was observed when comparing liver microsomes from individuals who had at least one UGT2B17 allele deleted [(+/0)+(0/0)] versus microsomes from UGT2B17 (+/+) subjects (P=0.004). When stratifying by the median value of NNAL-O-Gluc formation activity, a significantly (P=0.015) higher number of subjects with liver microsomes having low NNAL-O-Gluc formation activity contained the UGT2B17 null genotype compared to subjects with liver microsomes exhibiting high NNAL-O-Gluc formation activity. When stratifying by UGT2B7/UGT2B17 haplotypes, the association between the level of liver microsomal NNAL-O-glucuronide formation and increasing numbers of the UGT2B17 null allele was at the level of statistical significance for subjects with the UGT2B7 (*1/*2) (P=0.05) or UGT2B7 (*2/*2) (P<0.02) genotypes. These data suggest that the UGT2B17 deletion polymorphism is associated with a reduced rate of NNAL detoxification in vivo and may increase individual susceptibility to tobacco-related cancers.

Correlation between UDP-glucuronosyltransferase genotypes and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone glucuronidation phenotype in human liver microsomes.

The nicotine-derived tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, is one of the most potent and abundant procarcinogens found in tobacco and tobacco smoke, and glucuronidation of its major metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), is an important mechanism for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone detoxification. Substantial interindividual variability in urinary NNAL glucuronide formation has been observed in smokers and tobacco chewers. To determine whether genetic variations may play a role in this interindividual variability, NNAL-glucuronidating activities were analyzed in 78 human liver microsomal specimens and compared with the prevalence of missense polymorphisms in the two major NNAL-glucuronidating enzymes UGT1A4 and UGT2B7. In vitro assays using liver microsomal specimens from individual subjects demonstrated a 70- and 50-fold variability in NNAL-N-Gluc and NNAL-O-Gluc formation, respectively, and a 20-fold variability in the ratio of NNAL-N-Gluc:NNAL-O-Gluc formation. Microsomes from subjects with a homozygous polymorphic UGT1A4(24Thr)/UGT1A4(24Thr) genotype exhibited a significantly higher (P < 0.05) level of NNAL-N-Gluc activity compared with microsomes from subjects with the wild-type UGT1A4(24Pro)/UGT1A4(24Pro) genotype, and a significantly higher (P < 0.05) number of subjects with liver microsomes having high NNAL-N-Gluc formation activity contained the UGT1A4(24Thr)/UGT1A4(24Thr) genotype. Microsomes from subjects with the homozygous polymorphic UGT2B7(268Tyr)/UGT2B7(268Tyr) genotype exhibited a significantly lower level (P < 0.025) of NNAL-O-Gluc activity when compared with microsomes from subjects with the wild-type UGT2B7(268His)/UGT2B7(268His) genotype, and a significantly (P < 0.05) higher number of subjects with liver microsomes having low NNAL-O-Gluc formation activity contained the UGT2B7(268Tyr)/UGT2B7(268Tyr) genotype. These data suggest that the UGT1A4 codon 24 and UGT2B7 codon 268 polymorphisms may be associated with altered rates glucuronidation and detoxification of NNAL in vivo.

Characterization of N-glucuronidation of the lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in human liver: importance of UDP-glucuronosyltransferase 1A4.

The nicotine-derived tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is one of the most potent and abundant procarcinogens found in tobacco and tobacco smoke and is considered to be a causative agent for several tobacco-related cancers. Glucuronidation of the major metabolite of NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), has been implicated as an important mechanism for NNK detoxification. To characterize NNAL metabolism by N-glucuronidation in humans, high-pressure liquid chromatography was used to detect glucuronide conjugates of NNAL formed in human liver microsomes in vitro. In addition to peaks corresponding to the O-glucuronides of NNAL (NNAL-O-Gluc), a second series of peaks were observed in human liver microsomes that were identified by liquid chromatography-mass spectrometry to be NNAL N-glucuronides (NNAL-N-Gluc). Microsomes prepared from liver specimens from individual subjects (n = 42) exhibited substantial variability in the levels of NNAL-N-Gluc (49-fold variability) and NNAL-O-Gluc (49-fold variability) formed in vitro. This variability was likely not due to differences in tissue quality, as substantial variability (5-fold) was also observed in the ratio of NNAL-N-Gluc/NNAL-O-Gluc formation, with a mean ratio of 1.7 in the 42 specimens. Liver microsomes from smokers (n = 14) exhibited no significant difference in the levels of either NNAL-N-Gluc or NNAL-O-Gluc formation, or in the ratio of NNAL-N-Gluc/NNAL-O-Gluc formation, as compared with liver microsomes from never smokers (n = 28). Overexpressed UDP-glucuronosyltransferase (UGT) 1A4 exhibited significant levels of N-glucuronidating activity (V(max)/K(m) = 3.11 microl. min(-1). g(-1)) in vitro; no NNAL-N-glucuronide formation was detected for the 11 other overexpressed UGT enzymes tested in these studies. These results demonstrate the importance of N-glucuronidation in the metabolism of NNAL and the role of UGT1A4 in this pathway.

Maternal dietary docosahexanoic acid content affects the rat pup auditory system.

Previous studies of the effects of dietary docosahexanoic acid (DHA), 22:6n3, on neurodevelopment have focused mainly on visual-evoked potentials and indices of visual activity, measures that may be confounded by effects on the retina rather than on neural pathways. We investigated the effect of pre- and postnatal maternal dietary DHA content on auditory brainstem conduction times (ABCTs), the appearance of the auditory startle reflex (ASR), and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) activity in brainstem homogenates. Timed pregnant dams were fed, beginning on day 2 of gestation and throughout lactation, a purified diet containing one of three levels of DHA (0, 1, or 3% of total fatty acids, or 0, 0.4 or 1.2% of total energy). On postnatal day (PND) 3, pups were randomly crossfostered within diet groups to minimize litter effects and culled to 10 per litter. Cerebrums and milk from culled pups stomachs were collected for lipid analysis. The timing of appearance of the ASR was determined between PND 10 through 14 and ABCTs were measured in pups on PND 24 and 31. Pups were sacrificed on PND 31 and cerebrums were removed. In each of two replicated studies, pups in the 1% DHA group weighed significantly less on PND 3 and they gained significantly less weight from PND 3 to 31 compared with pups in the 0 or 3% groups (p<0.01). The auditory studies were not conducted on the 1% DHA group since measures of auditory function are in part a function of somatic growth. The tissue fatty acid data for the 1% DHA group did not show unexpected findings. Higher dietary DHA was reflected in milk and pup cerebrums, and levels of arachidonic acid were inversely related to levels of DHA. In the pups of dams fed diets containing 3% versus 0% DHA, the ASR appeared significantly later (p<0.001) and the ABCTs were longer (p<0.05) on PND 31. CNPase activity levels were not different between the 0 and 3% DHA groups. This study demonstrated that the auditory brainstem response is sensitive for identifying effects of diet on neurodevelopment, and that diets supplemented with high levels of DHA may exert a negative influence on central nervous system development, potentially through effects on myelin. This study suggests the need for further studies of pre- and postnatal long chain polyunsaturated fatty acid dietary supplementation.

Characterization of benzo(a)pyrene-trans-7,8-dihydrodiol glucuronidation by human tissue microsomes and overexpressed UDP-glucuronosyltransferase enzymes.

UDP-glucuronosyltransferase (UGT)-mediated glucuronidation of benzo(a)pyrene-trans-7,8-dihydrodiol (BPD), precursor to the potent mutagen benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide, may be an important pathway in the detoxification of benzo(a)pyrene. To better characterize this pathway in humans, high-pressure liquid chromatography (HPLC) was used to detect glucuronide conjugates of BPD formed in vitro. Three peaks were detected by HPLC after incubation of racemic BPD with human liver microsomes; these were identified as monoglucuronides by liquid chromatography-mass spectrometry analysis. Proton nuclear magnetic resonance spectroscopy of isolated fractions, combined with HPLC analysis of the glucuronide products from human liver microsomal incubations with purified benzo(a)pyrene-trans-7S,8S-dihydrodiol [(+)-BPD] and benzo(a)pyrene-trans-7R,8R-dihydrodiol [(-)-BPD] forms of BPD, indicated that peak 1 contained the 7-glucuronide of 7S,8S-BPD (BPD-7S-Gluc), peak 2 was a mixture of the 7-glucuronide of 7R,8R-BPD (BPD-7R-Gluc) and the 8-glucuronide of 7S,8S-BPD (BPD-8S-Gluc), and peak 3 contained the 8-glucuronide of 7R, 8R-BPD (BPD-8R-Gluc). In liver microsomes, peak 1 (BPD-7S-Gluc) was the largest peak observed, whereas in microsomes from aerodigestive tract tissues, peak 2 (both BPD-7R-Gluc and BPD-8S-Gluc) was the largest HPLC peak observed. The liver enzymes UGT1A1 and UGT2B7 formed BPD-7S-Gluc as the major diastereomer, whereas UGT1A8 and UGT1A10, extrahepatic enzymes present in the aerodigestive tract, preferentially formed both BPD-7R-Gluc and BPD-8S-Gluc. In addition, both UGT1A9 and UGT1A7 preferentially formed BPD-7R-Gluc. No detectable glucuronidating activity against BPD was observed by UGT1A3, UGT1A4, UGT1A6, UGT2B4, UGT2B15, or UGT2B17. The affinity of individual UGT enzymes as determined by K(m) analysis was UGT1A10 > UGT1A9 > UGT1A1 > UGT1A7 for (-)-BPD and UGT1A10 > UGT1A9 > UGT2B7 approximately UGT1A1 > UGT1A7 for (+)-BPD. These results suggest that several UGTs may play an important role in the overall glucuronidation of BPD in humans, with UGT1A1, UGT1A7, UGT1A9, UGT1A10 and potentially UGT1A8 playing an important role in the glucuronidation of the procarcinogenic (-)-BPD enantiomer, and that the stereospecific activity exhibited by different UGTs against BPD is consistent with tissue-specific patterns of BPD glucuronide diastereomer formation and UGT expression.