Eribulin induces micronuclei and enhances the nuclear localization of cGAS in triple-negative breast cancer cells.

Eribulin (ERI), clinically utilized for locally advanced or metastatic breast tumors, has shown potential links to the immune system. Notably, the cGAS-STING pathway, a key component of innate immunity, has gained prominence. Yet, limited reports explore ERI's effects on the cGAS-STING pathway. Additionally, the nuclear presence of cGAS remains poorly understood. This study uniquely delves into ERI's impact on both the cytosolic cGAS-STING pathway and nuclear cGAS. ERI enhances nuclear localization of cGAS, resulting in hyper-activation of the cGAS-STING pathway in triple-negative breast cancer cells. Reduction of cGAS heightened both cell proliferation and ERI sensitivity. In clinical data using ERI in a neo-adjuvant setting, patients with low cGAS cases exhibited reduced likelihood of achieving pathological complete response after ERI treatment. These findings illuminate the potential of cGAS and IFNß as predictive biomarkers for ERI sensitivity, providing valuable insights for personalized breast cancer treatment strategies.

Eribulin induces micronuclei and enhances the nuclear localization of cGAS in triple-negative breast cancer cells.

Eribulin (ERI), clinically utilized for locally advanced or metastatic breast tumors, has shown potential links to the immune system. Notably, the cGAS-STING pathway, a key component of innate immunity, has gained prominence. Yet, limited reports explore ERI's effects on the cGAS-STING pathway. Additionally, the nuclear presence of cGAS remains poorly understood. This study uniquely delves into ERI's impact on both the cytosolic cGAS-STING pathway and nuclear cGAS. ERI enhances nuclear localization of cGAS, resulting in hyper-activation of the cGAS-STING pathway in triple-negative breast cancer cells. Reduction of cGAS heightened both cell proliferation and ERI sensitivity. In clinical data using ERI in a neo-adjuvant setting, patients with low cGAS cases exhibited reduced likelihood of achieving pathological complete response after ERI treatment. These findings illuminate the potential of cGAS and IFNß as predictive biomarkers for ERI sensitivity, providing valuable insights for personalized breast cancer treatment strategies.

Ex vivo dual gene therapy using human adipocytes secreting anti-HER2 antibody on HER2-positive xenograft tumor models.

BACKGROUND: Although targeted treatments against human epidermal growth factor receptor 2 (HER2) have improved survival in patients with metastatic HER2-positive breast cancer, long and repeated treatment is time-consuming and costly for patients. To reduce these burdens, we developed ex vivo gene-transduced adipocytes that secrete anti-HER2 antibodies and evaluated their anti-tumor effects. METHODS: Ceiling culture-derived proliferative adipocytes (ccdPA) secreting anti-HER2 antibody against domain IV receptors: TRA-ccdPA, and domain II receptors: PER-ccdPA, were constructed using a plasmid lentivirus. Delivery of secreted antibody and its specific binding to HER2 breast cancer were evaluated in vitro and in vivo. To optimize antibody production from ccdPA, different conditions of ccdPA implantation were examined. Anti-tumor efficacy was evaluated in HER2-positive-cancer-inoculated nude mice. RESULTS: Anti-HER2 antibody against domain II was identified in supernatants from PER-ccdPAs. The optimal method to achieve the highest concentration of antibody in mouse sera was injecting differentiated ccdPA cells into the mammary fat pad. Antibody in supernatants from PER-ccdPAs bound to the surface of HER2-positive breast cancer cells similar to pertuzumab. Antibodies in mouse sera were delivered to HER2-positive breast cancer tumors and tumor necrosis was observed microscopically. One-time administration of combined TRA-ccdPAs and PER-ccdPAs produced antibody continuously in mouse sera, and anti-tumor effects were maintained for the duration of this study in xenograft models. Furthermore, combination therapy significantly suppressed tumor growth compared with a single administration. CONCLUSION: Ex vivo gene-transduced adipocytes might be useful for cell-based gene therapy. This system may be a platform for various antibody therapies.

Less necessity of adjuvant S-1 treatment in non-monarchE-eligible patients.

BACKGROUND: In monarchE and Postoperative Therapy with Endocrine and TS-1 (POTENT) trials, abemaciclib and S-1 have, respectively, shown to be effective as adjuvant therapies for luminal breast cancer (BC), although whether patients who meet the criteria are at high risk of recurrence compared to non-eligible patients is still unknown. Here, we investigated recurrence risk according to the criteria of each trial in Japanese patients. METHODS: We reviewed the records of 992 patients who received surgery at Chiba University Hospital for stage I-III BC from January 2017 to May 2022 and selected 553 analytic cohort patients and retrospectively analyzed the relapse-free survival of the patients as the primary endpoint. High-recurrence risk was defined according to monarchE trial and POTENT trial. RESULTS: The 5-year RFS for monarchE cohort 1 and cohort 2 eligible patients were 77.78% and 89.33%, respectively, which were significantly lower than monarchE non-eligible patients (98.31%; p < 0.0001). However, the 5-year RFS rate for POTENT eligible patients (90.51%) was lower than for POTENT non-eligible patients (98.75%; p = 0.0001); excluding those who met the monarchE criteria, the prognosis of POTENT eligible patients had no significant differences from the prognosis of patients with POTENT non-eligible BC (p = 0.3100). CONCLUSION: MonarchE criteria accurately identify patients who are prone to relapse. Moreover, although POTENT criteria also suggested a reasonable capacity for recurrence prediction, there was no significant difference in recurrence between POTENT non-eligible patients and the patients who were POTENT but not monarchE eligible. This might offer justification for reconsidering the use of S-1 in monarchE non-eligible patients.

Attenuation of growth hormone production at the fetal stage is critical for dioxin-induced developmental disorder in rat offspring.

Although dioxins and related chemicals have been suspected to disrupt child development, their toxic mechanism remains poorly understood. Our previous studies in rat fetuses revealed that maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a highly-toxic dioxin, suppresses fetal synthesis of pituitary growth hormone (GH) that is essential for development. This study examined the hypothesis that attenuating GH expression in fetuses triggers developmental disorders. Treating pregnant rats with 1 µg/kg TCDD reduced the circulating level of GH and its downstream factor, insulin-like growth factor-1 (IGF-1), in the offspring only during the fetal and early neonatal stages. Although maternal TCDD exposure resulted in low body weight and length at babyhood and defects in the learning and memory ability at adulthood, GH supplementation in TCDD-exposed fetuses restored or tended to restore the defects including IGF-1 downregulation. Moreover, maternal TCDD exposure decreased the number of GH-positive cells during the fetal/neonatal stage. A microarray analysis showed that TCDD reduced the expression of death-associated protein-like 1 (DAPL1), a cell cycle-dependent proliferation regulator, in the fetal pituitary gland. In addition, TCDD treatment attenuated proliferating cells and cyclin mRNA expression in the fetal pituitary gland. Aryl hydrocarbon receptor (AHR)-knockout fetuses were insensitive to TCDD treatment, indicating that the TCDD-induced reduction in DAPL1 and GH mRNAs expression was due to AHR activation. Finally, DAPL1 knockdown suppressed GH and cyclin D2 expression in fetal pituitary cells. These results provide a novel evidence that dioxin suppresses GH-producing cell proliferation and GH synthesis due to partly targeting DAPL1, thereby impairing offspring development.

Gestational dioxin exposure suppresses prolactin-stimulated nursing in lactating dam rats to impair development of postnatal offspring.

A number of epidemiological studies have implicated environmental chemicals including dioxins in the induction of negative effects on child development. To clarify the underlying mechanisms, almost all toxicologists have concentrated on effects on the offspring themselves. We examined an alternative hypothesis that gestational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a highly-toxic dioxin, targets factors related to maternal childcare to disturb offspring development. Oral administration of TCDD (1 µg/kg) to pregnant rats on gestational day 15 suppressed maternal licking behavior, a nursing behavior, and mammary gland maturation during the lactational stage, as well as the body weight and short-term memory of postnatal offspring. In support of these findings, maternal production of prolactin, a pituitary hormone essential for nursing including milk production, was decreased during the same period. Intracerebroventricular infusion of prolactin to dioxin-exposed dams restored or tended to restore many of the above defects observed both in mothers and offspring. The TCDD-dependent defects in maternal nursing behaviors can be due to a direct action on aryl hydrocarbon receptor (AHR) of lactating dams, because they did not emerge in AHR-knockout dams or control dams with TCDD-exposed offspring. Further examinations revealed that TCDD induces transforming growth factor ß1 expression, which suppresses prolactin-producing cell proliferation, in a nursing period-specific manner. In agreement with this, the number of prolactin-positive cells in nursing dams was decreased by TCDD. These results provide novel evidence that gestational dioxin exposure attenuates prolactin-stimulated nursing in lactating dams to impair offspring development, and that immaturity of prolactin-producing cells can contribute to them.

[A Case of Synchronous Colorectal Cancer Successfully Treated by Laparoscopic Surgery with Two Segmental Anastomoses].

A 63-year-old man was admitted for the evaluation of Hb 4.8 g/dL anemia. He underwent colon fiberscopy and was subsequently diagnosed with synchronous cancers of the ascending colon and rectum. He underwent laparoscopic ileocecal resection and low anterior resection with 2 segmental anastomoses. The histopathological diagnosis of A/C and rectal cancer was Stage Ⅱ and Stage Ⅲa, respectively. His treatment was completed after 6months of adjuvant chemotherapy with oral TS-1, which was followed by a subsequent 2 year follow-up study, without disease recurrence. Operations of synchronous cancers with 2 segmental anastomoses usually require longer surgical time and are associated with more postoperative complications compared with a single segmental anastomosis. We report a case of synchronous colorectal cancer successfully treated by laparoscopic surgery with 2 segmental anastomoses.

UDP-Glucuronosyltransferase (UGT)-mediated attenuations of cytochrome P450 3A4 activity: UGT isoform-dependent mechanism of suppression.

BACKGROUND AND PURPOSE: Cytochrome P450 (CYP, P450) 3A4 is involved in the metabolism of 50% of drugs and its catalytic activity in vivo is not explained only by hepatic expression levels. We previously demonstrated that UDP-glucuronosyltransferase (UGT) 2B7 suppressed CYP3A4 activity through an interaction. In the present study, we target UGT1A9 as another candidate modulator of CYP3A4. EXPERIMENTAL APPROACH: We prepared co-expressed enzymes using the baculovirus-insect cell expression system and compared CYP3A4 activity in the presence and absence of UGT1A9. Wistar rats were treated with dexamethasone and liver microsomes were used to elucidate the role of CYP3A-UGT1A interactions. KEY RESULTS: UGT1A9 and UGT2B7 interacted with and suppressed CYP3A4. Kinetic analyses showed that both of the UGTs significantly reduced Vmax of CYP3A4 activity. In addition, C-terminal truncated mutants of UGT1A9 and UGT2B7 still retained the suppressive capacity. Dexamethasone treatment induced hepatic CYP3As and UGT1As at different magnitudes. Turnover of CYP3A was enhanced about twofold by this treatment. CONCLUSION AND IMPLICATIONS: The changes of kinetic parameters suggested that UGT1A9 suppressed CYP3A4 activity with almost the same mechanism as UGT2B7. The luminal domain of UGTs contains the suppressive interaction site(s), whereas the C-terminal domain may contribute to modulating suppression in a UGT isoform-specific manner. CYP3A-UGT1A interaction seemed to be disturbed by dexamethasone treatment and the suppression was partially cancelled. CYP3A4-UGT interactions would help to better understand the causes of inter/intra-individual differences in CYP3A4 activity.

Elevation of endocannabinoids in the brain by synthetic cannabinoid JWH-018: mechanism and effect on learning and memory.

The impairment of learning and memory is a well-documented effect of both natural and synthetic cannabinoids. In the present study, we aimed to investigate the effect of acute administration of JWH-018, a synthetic cannabinoid, on the hippocampal metabolome to assess biochemical changes in vivo. JWH-018 elevated levels of the endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG). The increase of endocannabinoid levels in response to JWH-018 could be inhibited by co-administration of AM251, a CB1 receptor antagonist. Biochemical analyses revealed that this was the result of suppression of two hydrolases involved in endocannabinoid degradation (fatty acid amide hydrolase [FAAH] and monoacylglycerol lipase [MAGL]). Additionally, we showed that JWH-018 causes a reduction in the levels of brain-derived neurotrophic factor (BDNF), which is known to modulate synaptic plasticity and adaptive processes underlying learning and memory. The decrease of BDNF following JWH-018 treatment was also rescued by co-administration of AM251. As both endocannabinoids and BDNF have been shown to modulate learning and memory in the hippocampus, the alteration of their levels in response to JWH-018 may explain the contribution of synthetic cannabinoids to impairment of memory.

[A Case of Pancreatic Metastasis from Colon Cancer Successfully Treated with Capecitabine/Oxaliplatin plus Bevacizumab].

A 50-year-old man underwent low anterior resection for rectal cancer. The final diagnosis was rectal cancer of pT3N0M0, fStage Ⅱ. CT performed for examination of obstructive jaundice at 17 months after surgery revealed metastatic lesions of the pancreatic head and right lung. By core needle biopsies, the lesions were pathologically diagnosed as metachronous metastases of rectal cancer. Chemotherapy was carried out but was discontinued at 5 courses due to severe side effects. The pancreatic metastasis disappeared after 11 months. As the lung metastasis remained, a right upper lobectomy was performed 1 month later. The patient remains alive without recurrence 6 months after the partial lung resection.

[A Case of Laparoscopic Gastrojejunostomy for Duodenal Obstruction Caused by Unresectable Locally Advanced Pancreatic Cancer].

The patient was an 85-year-old man who received chemotherapy with gemcitabine for 2 years 9 months under the diagnosis of unresectable locally advanced pancreatic body and tail cancer. He visited our hospital because of anorexia, upper abdominal fullness, and vomiting. A CT scan showed severe stenosis in the third portion of the duodenum, which was associated with the direct invasion of the advanced pancreatic cancer. Upper gastrointestinal fiberscopy revealed a severe duodenal obstruction; however, pancreatic cancer exposure within the duodenal mucosa was not observed. As the stenosis of the duodenum was relatively smooth because of the cancer invasion into only the submucosa, deviation of the metallic stent was possible, so we performed laparoscopic gastrojejunostomy. We started the surgery with 5-port settings. A slit was made in the gastric body by using ENDO-GIA®, and bypass surgery with a Roux-en-Y anastomosis was performed. The postoperative course was good, and oral intake resumed on the third postoperative day. Thereafter, he could leave the hospital with good progress and received systemic chemotherapy using gemcitabine. In the present case, an extramural gastrointestinal stenosis without cancer that was not exposed in the gastrointestinal mucosa was poorly fixed with gastrointestinal metallic stents and use of a deviating metallic stent was reported, so we chose laparoscopic gastrojejunostomy. In addition, after undergoing laparoscopic surgery, which is a minimally invasive treatment, he recovered quickly and shifted early to systemic chemotherapy. Herein, the usefulness of laparoscopic gastrojejunostomy for extramural stenosis is reported with a review of related literature.

[A Case of Extraosteosarcoma in the Omentum].

A 51-year-old female presented to our hospital with a chief complaint of abdominal pain. A blood test showed high ALP value(7,001 IU/L), and the abdominal CT showed a mass lesion of 20 cm in diameter with calcification and infiltrated surroundings. From these findings, we diagnosed the patient with peritonealcancer pre-operatively. The intraoperative findings showed an advanced tumor infiltrated into the sigmoid, transverse colon, small intestine and uterus. There were multiple suspected metastasis tumors in the peritonealcavity. Therefore, we resected the tumor as much as possible without curative surgery. Pathologically, the spindle cells were growing with bone formation. Immunostaining showed negative epithelial markers. The tumor protruded out of the intestinal wall, and the patient was diagnosed with extraskeletal osteosarcoma in the omentum. Chemotherapy with doxorubicin and cisplatin was initiated. Because of the disease progression and the presence of side effects, the patient discontinued chemotherapy and died 4 months after the operation. Extraskeletal osteosarcoma is a very rare tumor with poor prognosis. We reported a case of extraskeletal osteosarcoma in the omentum and review the literature.

Alleviation of fatty liver in a rat model by enhancing N1-methylnicotinamide bioavailability through aldehyde oxidase inhibition.

Nonalcoholic fatty liver disease (NAFLD) has increased worldwide in recent years. NAFLD is classified into two types, nonalcoholic fatty liver (NAFL), with few complications, and nonalcoholic steatohepatitis (NASH), which leads to liver cirrhosis or cancer. This study was based on previous reports that N1-methylnicotinamide (MNA) can stabilise sirtuin 1 protein, leading to decreased lipid levels in the liver. We hypothesised that fatty liver improvement by MNA would be further enhanced by suppressing its rapid metabolism by aldehyde oxidase in the liver. To test this, hydralazine (HYD), a potent aldehyde oxidase inhibitor, was administered orally to NAFL model rats. Liver triglyceride (TG) levels in the model were nearly unchanged by administration of MNA alone. In contrast, TG levels were marked decreased in NAFL rats treated with a combination of MNA and HYD. In addition, TG levels were decreased even in NAFL rats treated with only HYD. These findings supported our hypothesis that maintaining MNA concentrations in the liver, by suppressing MNA metabolism, would at least partially ameliorate fatty liver.

The aryl hydrocarbon receptor is indispensable for dioxin-induced defects in sexually-dimorphic behaviors due to the reduction in fetal steroidogenesis of the pituitary-gonadal axis in rats.

Many forms of the toxic effects produced by dioxins and related chemicals take place following activation of the aryl hydrocarbon receptor (AHR). Our previous studies have demonstrated that treating pregnant rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a highly toxic dioxin, attenuates the pituitary expression of gonadotropins to reduce testicular steroidogenesis during the fetal stage, resulting in the impairment of sexually-dimorphic behaviors after the offspring reach maturity. To investigate the contribution of AHR to these disorders, we examined the effects of TCDD on AHR-knockout (AHR-KO) Wistar rats. When pregnant AHR-heterozygous rats were given an oral dose of 1 µg/kg TCDD at gestational day (GD) 15, TCDD reduced the expression of pituitary gonadotropins and testicular steroidogenic proteins in male wild-type fetuses at GD20 without affecting body weight, sex ratio and litter size. However, the same defect did not occur in AHR-KO fetuses. Further, fetal exposure to TCDD impaired the activity of masculine sexual behavior after reaching adulthood only in the wild-type offspring. Also, in female offspring, not only the fetal gonadotropins production but also sexual dimorphism, such as saccharin preference, after growing up were suppressed by TCDD only in the wild-type. Interestingly, in the absence of TCDD, deleting AHR reduced masculine sexual behavior, as well as fetal steroidogenesis of the pituitary-gonadal axis. These results provide novel evidence that 1) AHR is required for TCDD-produced defects in sexually-dimorphic behaviors of the offspring, and 2) AHR signaling plays a role in gonadotropin synthesis during the developmental stage to acquire sexual dimorphism after reaching adulthood.

[A Case of Locally Recurrent Tumor Resected by Left Hemicolectomy with Left Nephrectomy Six Years after Curative Operation for Sigmoid Colon Cancer].

A 65-year-old woman underwent high anterior resection for sigmoid colon cancer in 2011. The histopathological diagnosis was type 2, 25×27 mm, tub2, SE, N2, ly2, v1, and Stage Ⅲb. Her treatment was completed after 6months of adjuvant chemotherapy with UFT plus UZEL followed by a 5-year follow-up study, without recurrence. However, 6years after the initial operation, a routine chest and abdominal CT scan showed a 24mm local recurrence involving the left urinary tract and bilateral lung lesions. Eight courses of systemic chemotherapy using FOLFOX plus panitumumab regimen was administered immediately. CT scan after chemotherapy showed that all masses were downsized and no new lesions were identified. We resected the recurrent tumor after considering it feasible by left hemicolectomy with left nephrectomy. Histopathological examination of the recurrent tumor revealed adenocarcinoma, consistent with that of the previous primary sigmoid colon cancer. She is currently undergoing systemic chemotherapy using the FOLFOX regimen. There has been no change in the lung lesions and no new lesions have developed. This is a very rare case of recurrence more than 5 years after curative resection of Stage Ⅲ colon cancer. This paper presents the case considering that keeping the patient under surveillance for more than 5 years enabled the disclosure of recurrence without subjective symptoms.

Dioxin-induced increase in leukotriene B4 biosynthesis through the aryl hydrocarbon receptor and its relevance to hepatotoxicity owing to neutrophil infiltration.

Dioxin and related chemicals alter the expression of a number of genes by activating the aryl hydrocarbon receptors (AHR) to produce a variety of disorders including hepatotoxicity. However, it remains largely unknown how these changes in gene expression are linked to toxicity. To address this issue, we initially examined the effect of 2,3,7,8-tetrachrolodibenzo-p-dioxin (TCDD), a most toxic dioxin, on the hepatic and serum metabolome in male pubertal rats and found that TCDD causes many changes in the level of fatty acids, bile acids, amino acids, and their metabolites. Among these findings was the discovery that TCDD increases the content of leukotriene B4 (LTB4), an inducer of inflammation due to the activation of leukocytes, in the liver of rats and mice. Further analyses suggested that an increase in LTB4 comes from a dual mechanism consisting of an induction of arachidonate lipoxygenase-5, a rate-limiting enzyme in LTB4 synthesis, and the down-regulation of LTC4 synthase, an enzyme that converts LTA4 to LTC4. The above changes required AHR activation, because the same was not observed in AHR knock-out rats. In agreement with LTB4 accumulation, TCDD caused the marked infiltration of neutrophils into the liver. However, deleting LTB4 receptors (BLT1) blocked this effect. A TCDD-produced increase in the mRNA expression of inflammatory markers, including tumor-necrosis factor and hepatic damage, was also suppressed in BLT1-null mice. The above observations focusing on metabolomic changes provide novel evidence that TCDD accumulates LTB4 in the liver by an AHR-dependent induction of LTB4 biosynthesis to cause hepatotoxicity through neutrophil activation.

Comprehensive Characterization of Mouse UDP-Glucuronosyltransferase (Ugt) Belonging to the Ugt2b Subfamily: Identification of Ugt2b36 as the Predominant Isoform Involved in Morphine Glucuronidation.

UDP-Glucuronosyltransferases (UGTs) are classified into three subfamilies in mice: Ugt1a, 2b, and 2a. In the Ugt1a subfamily, Ugt1a1 and 1a6 appear to correspond to human UGT1A1 and 1A6 The mouse is an important animal for its use in investigations, but the substrate specificities of Ugt isoforms belonging to the 2b subfamily in mice remain largely unknown. To address this issue, we characterized the substrate specificity of all isoforms of the Ugt2b subfamily expressed in the mouse liver. The cDNAs of Ugt1a1, Ugt2a3, and all the Ugt2b isoforms expressed in the liver were reverse-transcribed from the total RNA of male FVB-mouse livers and then amplified. A baculovirus-Sf9 cell system for expressing each Ugt was established. Of all the Ugts examined, Ugt2b34, 2b36, and 2b37 exhibited the ability to glucuronidate morphine with Ugt2b36, the most active in this regard. Ugt1a1, but also Ugt2b34, 2b36, and 2b37 to a lesser extent, preferentially catalyzed the glucuronidation of 17ß-estradiol on the 3-hydroxyl group (E3G). With these isoforms, E3G formation by Ugt1a1 was efficient; however, Ugt2b5 exhibited a preference for the 17ß-hydroxyl group (E17G). Ugt2b1 and Ugt2a3 formed comparable levels of E3G and E17G. Ugt2b1 and 2b5 were the only isoforms involved in chloramphenicol glucuronidation. As Ugt2b36 is highly expressed in the liver, it is most likely that Ugt2b36 is a major morphine Ugt in mouse liver. Regarding E3G formation, Ugt1a1, like the human homolog, seems to play an important role in the liver.

Metabolomic profiling of brain tissues of mice chronically exposed to heroin.

The chronic neurotoxicity of heroin on the nervous system is poorly understood. To address this issue, we comprehensively assessed the alteration of brain metabolomics caused by chronic heroin exposure and the withdrawal of heroin. Male C57BL/6J mice (n = 10) were given heroin (15 µmol/kg, i.p., twice a day) for 12 days while the withdrawal group received saline-treatment instead of heroin for the last two days. The control group received saline. We developed an UPLC-TOF/MS-based metabolomic approach to analyze the metabolites and carry out a metabolic pathway analysis in the brain. The major metabolites contributing to the discrimination were identified as amino acids, tricarboxylic-acid cycle intermediates, neurotransmitters, nucleotides and other compounds. A marked reduction in histidine and a slight but significant increase in phenylalanine and tryptophan were observed after heroin was withdrawn while the increased level of catecholamines was restored to baseline. Interestingly, N-acetylserotonin - a precursor of melatonin - was increased with the withdrawal of heroin while melatonin was markedly reduced along with the sub-chronic exposure to heroin. This shows that heroin disrupts not only the energy metabolism but also the biosynthesis of both catecholamines and melatonin in the mouse brain. Therefore, these substances are candidate biomarkers for chronic heroin-abuse.

α-Lipoic acid potentially targets AMP-activated protein kinase and energy production in the fetal brain to ameliorate dioxin-produced attenuation in fetal steroidogenesis.

Our previous studies demonstrated that treating pregnant rats with dioxins, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), targets the pituitary expression of luteinizing hormone (LH) to attenuate testicular steroidogenesis in fetuses, resulting in the imprinting of sexual immaturity of the offspring after reaching maturity. Furthermore, we found that although TCDD disturbs the tricarboxylic acid (TCA) cycle in the fetal hypothalamus, maternal co-treatment with α-lipoic acid (α-LA), a cofactor of the TCA cycle, restores a TCDD-produced reduction in the LH-evoked steroidogenesis as well as the TCA cycle activity in fetuses. However, the mechanism underlying the beneficial effect of α-LA remains to be fully elucidated. To address this issue, we compared the effect of α-LA with that of thiamine, another cofactor of the TCA cycle. As with α-LA, supplying thiamine to dams exposed to TCDD alleviates the reduced level of not only hypothalamic ATP but also pituitary LH and testicular steroidogenic protein in fetuses. However, thiamine had a much weaker effect than α-LA. In agreement with ATP attenuation, TCDD activated AMP-activated protein kinase (AMPK), a negative regulator of LH production, whereas the supplementation of α-LA allowed recovery from this defect. Furthermore, α-LA restored the TCDD-produced reduction in the pituitary expression of the receptor for gonadotropin-releasing hormone (GnRH), an upstream regulator of LH synthesis. These results suggest that α-LA rescues TCDD-produced attenuation during fetal steroidogenesis due not only to facilitation of energy production through the TCA cycle but also through suppression of AMPK activation, and the pituitary GnRH receptor may serve as a mediator of these effects.

Introduction of an N-Glycosylation Site into UDP-Glucuronosyltransferase 2B3 Alters Its Sensitivity to Cytochrome P450 3A1-Dependent Modulation.

Our previous studies have demonstrated functional protein-protein interactions between cytochrome P450 (CYP) 3A and UDP-glucuronosyltransferase (UGT). However, the role of carbohydrate chains of UGTs in the interaction with CYP is not well understood. To address this issue, we examined whether CYP3A1 modulates the function of UGT2B3 which lacks potential glycosylation sites. We also examined whether the introduction of N-glycosylation to UGT2B3 affects CYP3A-dependent modulation of UGT function. To introduce a potential glycosylation site into UGT2B3, Ser 316 of UGT2B3 was substituted with Asn by site-directed mutagenesis. A baculovirus-Sf-9 cell system for expressing CYP3A1 and UGT2B3/UGT2B3(S316N) was established using a Bac-to-Bac system. Glycosylation of UGT2B3(S316N) was demonstrated in this expression system. The microsomal activity of recombinant UGT was determined using 4-methylumbelliferone as a substrate. The effect of CYP3A1 co-expression on UGT function was examined by comparing the kinetic profiles between single (UGT alone) and double expression (UGT plus CYP) systems. The kinetics of the two expression systems fitted a Michaelis-Menten equation. When the 4-MU concentration was varied, co-expression of CYP3A1 lowered the Vmax of UGT2B3-mediated conjugation. Conversely, for UGT2B3(S316N), the Vmax in the dual expression system was higher than that in the single expression system. The data obtained demonstrate that the introduction of N-glycosylation to UGT2B3 alters its sensitivity to CYP3A1-dependent modulation while CYP3A1 enhanced UGT2B3(S316N) activity, and wild-type UGT2B3 was suppressed by CYP3A1. These data suggest that N-glycosylation of UGT is one of the determinants regulating the interaction between CYP3A and UGT.