Use of mixed-effect models and tolerance limits to evaluate control cynomolgus monkey body weight change and variability during preclinical toxicology studies.

Cynomolgus monkeys are an important and widely used species in preclinical toxicology studies. During the in-life phase of study, body weight effects may be indicative of toxicity; however, trends in body weight and body weight variability are often difficult to interpret due to small sample size and/or inter- and intra-animal variability. The present analysis utilizes mixed-effect modelling, which incorporates random and fixed effects into linear regression models, to evaluate control monkey body weight trends and variability relative to baseline (initial) weight and study duration. The primary aim of this analysis is to evaluate whether mixed-effect model based tolerance limits can aide in determining whether apparent test article-related changes in body weight deviate more than the predicted variability defined by the model tolerance limits. The models for this study are based on vehicle control animal body weight data from the following studies: 1-month (20 studies, 198 animals), 3-month (19 studies, 180 animals), and 9-month (17 studies, 182 animals). The analysis presented herein provides the framework for evaluating control monkey body weight change in studies with small sample size, and anticipated control monkey body weight change relative to gender and study duration.

Nuclear factor erythroid 2-related factor 2 deletion impairs glucose tolerance and exacerbates hyperglycemia in type 1 diabetic mice.

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) induces a battery of cytoprotective genes after oxidative stress. Nrf2 aids in liver regeneration by altering insulin signaling; however, whether Nrf2 participates in hepatic glucose homeostasis is unknown. Compared with wild-type mice, mice lacking Nrf2 (Nrf2-null) have lower basal serum insulin and prolonged hyperglycemia in response to an intraperitoneal glucose challenge. In the present study, blood glucose, serum insulin, urine flow rate, and hepatic expression of glucose-related genes were quantified in male diabetic wild-type and Nrf2-null mice. Type 1 diabetes was induced with a single intraperitoneal dose (200 mg/kg) of streptozotocin (STZ). Histopathology and serum insulin levels confirmed depleted pancreatic beta-cells in STZ-treated mice of both genotypes. Five days after STZ, Nrf2-null mice had higher blood glucose levels than wild-type mice. Nine days after STZ, polyuria occurred in both genotypes with more urine output from Nrf2-null mice (11-fold) than wild-type mice (7-fold). Moreover, STZ-treated Nrf2-null mice had higher levels of serum beta-hydroxybutyrate, triglycerides, and fatty acids 10 days after STZ compared with wild-type mice. STZ reduced hepatic glycogen in both genotypes, with less observed in Nrf2-null mice. Increased urine output and blood glucose in STZ-treated Nrf2-null mice corresponded with enhanced gluconeogenesis (glucose-6-phosphatase and phosphoenolpyruvate carboxykinase)- and reduced glycolysis (pyruvate kinase)-related mRNA expression in their livers. Furthermore, the Nrf2 activator oltipraz lowered blood glucose in wild-type but not Nrf2-null mice administered STZ. Collectively, these data indicate that the absence of Nrf2 worsens hyperglycemia in type I diabetic mice and Nrf2 may represent a therapeutic target for reducing circulating glucose levels.

Enhanced expression of Nrf2 in mice attenuates the fatty liver produced by a methionine- and choline-deficient diet.

Oxidative stress has been proposed as an important promoter of the progression of fatty liver diseases. The current study investigates the potential functions of the Nrf2-Keap1 signaling pathway, an important hepatic oxidative stress sensor, in a rodent fatty liver model. Mice with no (Nrf2-null), normal (wild type, WT), and enhanced (Keap1 knockdown, K1-kd) expression of Nrf2 were fed a methionine- and choline-deficient (MCD) diet or a control diet for 5 days. Compared to WT mice, the MCD diet-caused hepatosteatosis was more severe in the Nrf2-null mice and less in the K1-kd mice. The Nrf2-null mice had lower hepatic glutathione and exhibited more lipid peroxidation, whereas the K1-kd mice had the highest amount of glutathione in the liver and developed the least lipid peroxidation among the three genotypes fed the MCD diet. The Nrf2 signaling pathway was activated by the MCD diet, and the Nrf2-targeted cytoprotective genes Nqo1 and Gstalpha1/2 were induced in WT and even more in K1-kd mice. In addition, Nrf2-null mice on both control and MCD diets exhibited altered expression profiles of fatty acid metabolism genes, indicating Nrf2 may influence lipid metabolism in liver. For example, mRNA levels of long chain fatty acid translocase CD36 and the endocrine hormone Fgf21 were higher in livers of Nrf2-null mice and lower in the K1-kd mice than WT mice fed the MCD diet. Taken together, these observations indicate that Nrf2 could decelerate the onset of fatty livers caused by the MCD diet by increasing hepatic antioxidant and detoxification capabilities.

Circadian expression profiles of drug-processing genes and transcription factors in mouse liver.

Temporal coordination of hepatic drug-processing gene (DPG) expression facilitates absorption, biotransformation, and excretion of exogenous and endogenous compounds. To further elucidate the circadian rhythm of hepatic DPG expression, male C57BL/6 mice were subjected to a standard 12-h light/dark cycle, and livers were collected at 2:00, 6:00, and 10:00 AM and 2:00, 6:00, and 10:00 PM. The mRNAs of hepatic phase I enzymes (cytochromes P450, aldehyde dehydrogenases, and carboxylesterases), phase II enzymes (glucuronosyltransferases, sulfotransferases, and glutathione S-transferases), uptake and efflux transporters, and transcription factors were quantified. Messenger RNAs of various genes were graphed across time of day and compared by hierarchical clustering. In general, the mRNA of phase I enzymes increased during the dark phase, whereas the mRNAs of most phase II enzymes and transporters reached maximal levels during the light phase. The majority of hepatic transcription factors exhibited expression peaks either before or after the onset of the dark phase. During the same time period, the negative clock regulator gene Rev-Erbalpha and the hepatic clock-controlled gene Dbp also reached mRNA expression peaks. Considering their important role in xenobiotic metabolism, hepatic transcription factors, such as constitutive androstane receptor, pregnane X receptor, aryl hydrocarbon receptor, and peroxisomal proliferator activated receptor alpha, may be involved in coupling the hepatic circadian clock to environmental cues. Taken together, these data demonstrate that the circadian expression of the DPG battery and transcription factors contribute to the temporal detoxification cycle in the liver.

Potential mechanisms of target-independent uptake and toxicity of antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are a promising therapeutic modality for oncology indications. The concept of an ADC platform is to increase the therapeutic index (TI) of chemotherapeutics through more selective delivery of cytotoxic agents to tumor cells while limiting exposure to healthy normal cells. Despite the use of antibodies targeting antigens abundantly and/or exclusively expressed on cancer cells (i.e., target cells), dose limiting toxicities (DLTs) in normal cells/tissues are frequently reported even at suboptimal therapeutic doses. Although advancement of ADC technology has helped to optimize all three key components (i.e., mAb, linker, and payload), DLTs remain a key challenge for ADC development. Mechanisms of ADC toxicity in normal cells/tissues are not clearly understood, but the majority of DLTs are considered to be target-independent. In addition to linker-drug instability contributing to the premature release of cytotoxic drug (payload) in circulation, uptake/trafficking of intact ADCs by both receptor-dependent (FcγRs, FcRn and C-type lectin receptors), and-independent (non-specific endocytosis) mechanisms may contribute to off-target toxicity in normal cells. In this article, we review potential mechanisms of target-independent ADC uptake and toxicity in normal cells, as well as discuss components of ADCs which may influence these mechanisms. This information will provide a deeper understanding of the underlying mechanisms of ADC off-target toxicity and prove helpful toward improving the overall TI of the next generation of ADCs.

NF-E2-related factor 2 inhibits lipid accumulation and oxidative stress in mice fed a high-fat diet.

NF-E2-related factor 2 (Nrf2) is a transcription factor that is activated by oxidative stress and electrophiles that regulates the expression of numerous detoxifying and antioxidant genes. Previous studies have shown that Nrf2 protects the liver from xenobiotic toxicity; however, whether Nrf2 plays a role in lipid homeostasis in liver is not known. Accordingly, wild-type and Nrf2-null mice were fed a high-fat diet (HFD) for up to 4 weeks. Hepatic gene expression and lipid profiles were analyzed for changes in fatty acid, triglyceride, and cholesterol status. It is interesting to note that HFD reduced the mRNA expression of Nrf2 and its target genes in wild-type mice. The mRNA expression of lipogenic and cholesterologenic transcriptional factors and their target genes, such as sterol regulatory element-binding proteins 1c and 2, fatty acid synthase, acetyl-CoA carboxylase 1, fatty acid elongase, 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase, and low-density lipoprotein receptor mRNA expression were higher in Nrf2-null mice compared with wild-type mice after feeding a HFD, suggesting that Nrf2 may suppress these pathways. Hepatic triglycerides and cholesterol levels were not different between genotypes, whereas concentrations of hepatic free fatty acid and malondialdehyde equivalents were higher in Nrf2-null mice compared with wild-type mice 4 weeks after HFD feeding. Overall, these results suggest that Nrf2 inhibits lipid accumulation and oxidative stress in mouse liver after feeding a HFD, probably by interfering with lipogenic and cholesterologenic pathways.

Attenuation of TCDD-induced oxidative stress by 670 nm photobiomodulation in developmental chicken kidney.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a potent developmental teratogen inducing oxidative stress and sublethal changes in multiple organs, provokes developmental renal injuries. In this study, we investigated TCDD-induced biochemical changes and the therapeutic efficacy of photobiomodulation (670 nm; 4 J/cm(2)) on oxidative stress in chicken kidneys during development. Eggs were injected once prior to incubation with TCDD (2 pg/g or 200 pg/g) or sunflower oil vehicle control. Half of the eggs in each dose group were then treated with red light once per day through embryonic day 20 (E20). Upon hatching at E21, the kidneys were collected and assayed for glutathione peroxidase, glutathione reductase, catalase, superoxide dimutase, and glutathione-S-transferase activities, as well as reduced glutathione and ATP levels, and lipid peroxidation. TCDD exposure alone suppressed the activity of the antioxidant enzymes, increased lipid peroxidation, and depleted available ATP. The biochemical indicators of oxidative and energy stress in the kidney were reversed by daily phototherapy, restoring ATP and glutathione contents and increasing antioxidant enzyme activities to control levels. Photobiomodulation also normalized the level of lipid peroxidation increased by TCDD exposure. The results of this study suggest that 670 nm photobiomodulation may be useful as a noninvasive treatment for renal injury resulting from chemically induced cellular oxidative and energy stress.

Melatonin as a principal component of red light therapy.

Melatonin is well recognized for its role as a potent antioxidant and is directly implicated in the free radical theory of aging [1] [Reiter RJ, Pablos MI, Agapito TT, Guerrero JM. Melatonin in the context of the free radical theory of aging. Ann N Y Acad Sci 1996;786:362-78]. Moreover, melatonin has been shown to retard age-related increases in lipid peroxidation and oxidative damage [2] [Okatani Y, Wakatsuki A, Reiter RJ. Melatonin protects hepatic mitochondrial respiratory chain activity in senescence-accelerated mice. J Pineal Res 2002;32:143-8] and to act directly upon the immune system [3] [Poon AM, Liu ZM, Pang CS, Brown GM, Pang SF. Evidence for a direct action of melatonin on the immune system. Biol Signals 1994;3:107-17]. This report focuses on characterizing documented functions of melatonin in the context of red light therapy and proposes that melatonin is a potential mediator of red light's therapeutic effects, a hypothesis that is as yet untested. Red light therapy (670 nm, 4J/cm(2)) has been shown to restore glutathione redox balance upon toxicological insult and enhance both cytochrome c oxidase and energy production, all of which may be affected by melatonin. The red light treatment has also been successfully implemented in the clinical setting for its effectiveness in reducing both the number of incidences and severity of oral mucositis resulting in part from the chemotherapy and/or radiation administered prior to bone marrow transplants. Moreover, red light therapy improves wound healing and is being further tested for its ability to ameliorate toxicant-induced retinal and visual cortical neuron damage. Researchers in the growing field of light therapy may be in a position to draw from and collaborate with melatonin researchers to better characterize this alternative treatment.

External heart deformities in passerine birds exposed to environmental mixtures of polychlorinated biphenyls during development.

Necropsy-observable cardiac deformities were evaluated from 283 nestling passerines collected from one reference site and five polychlorinated biphenyl (PCB)-contaminated sites around Bloomington and Bedford, Indiana, USA. Hearts were weighed and assessed on relative scales in three dimensions (height, length, and width) and for externally visible deformities. Heart weights normalized to body weight (heart somatic index) were decreased significantly at the more contaminated sites in both house wren (Troglodytes aedon) and tree swallow (Tachycineta bicolor). Heart somatic indices significantly correlated with log PCB concentrations in Carolina chickadee (Parus carolinesis) and tree swallow and with log 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalent values in tree swallow alone. Ventricular length was increased significantly in eastern bluebirds (Sialia sialis) and decreased significantly in Carolina chickadee and tree swallow from contaminated sites versus the reference site. Heart length regressed significantly against the log PCB concentrations (Carolina chickadee and tree swallow) or the square of the PCB concentrations (red-winged blackbird [Agelaius phoeniceus]) in a sibling bird. The deformities that were observed most at the contaminated sites included abnormal tips (pointed, rounded, or flattened), center rolls, macro- and microsurface roughness, ventricular indentations on the ventral or dorsal surface, lateral ventricular notches, visibly thin ventricular walls, and changes in overall heart shape. A pooled heart deformity index regressed significantly against the logged contaminant concentrations for all species except red-winged blackbird. These results indicate that developmental changes in heart morphometrics and shape abnormalities are quantifiable and may be sensitive and useful indicators of PCB-related developmental impacts across many avian species.

Survivorship and mortality implications of developmental 670-nm phototherapy: dioxin co-exposure.

OBJECTIVE: We assessed the effect of 670-nm light therapy on dioxin-induced embryonic mortality in chickens (Gallus gallus). BACKGROUND DATA: Developmental photobiomodulation using 670-nm light-emitting diode (LED) arrays improves hatching success and increases body size in hatchling chickens. Photobiomodulation also stimulates signaling pathways resulting in improved energy metabolism, antioxidant production and cell survival. Dioxin causes embryonic mortality, including increases in the frequency of chicken embryos that pip but can't go to hatch. We hypothesized that 670-nm LED therapy would attenuate dioxin-induced embryo mortality. METHODS: Fertile chicken eggs were injected with control or 2, 20, or 200 ppt 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) prior to the start of incubation. Half of the eggs in each dose group were treated once per day from embryonic days 0-20 with 670-nm LED light at a fluence of 4 J/cm(2). In ovo survival and hatching success were compared between dose groups and LED treatment. RESULTS: LED therapy decreased the embryonic mortality rate by 41%, resulting in increased embryonic survival and improved hatching success in eggs exposed to 200 ppt dioxin. However, at sub-lethal dioxin concentrations and in oil-treated controls, LED therapy slightly increased mortality. CONCLUSION: Overall survivorship and hatching success of chicks developmentally exposed to dioxin concentrations above the lethality threshold (>100 ppt TCDD) is improved by 670-nm LED treatment administered throughout the gestation period, but the relationship may be complicated by an LED-oil interaction.

Brief report: embryonic growth and hatching implications of developmental 670-nm phototherapy and dioxin co-exposure.

OBJECTIVE: We assessed the effect of 670-nm light therapy on growth and hatching kinetics in chickens (Gallus gallus) exposed to dioxin. BACKGROUND DATA: Photobiomodulation has been shown to stimulate signaling pathways resulting in improved energy metabolism, antioxidant production, and cell survival. In ovo treatment with 670-nm light-emitting diode (LED) arrays improves hatching success and increases hatchling size in control chickens. Under conditions where developmental dioxin exposure is above the lethality threshold (100 ppt), phototherapy attenuates dioxin-induced early embryonic death. We hypothesized that 670-nm LED therapy would attenuate dioxin-induced developmental anomalies and increase hatching success. METHODS: Fertile chicken eggs were injected with control oil, 2, 20, or 200 ppt dioxin, or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) prior to the start of incubation. Half of the eggs in each dose group were treated once per day from embryonic days 0-20 with 670-nm LED light at a fluence of 4 J/cm2. Hatchling size, organ weights, and energy parameters were compared between dose groups and LED treatment. RESULTS: LED therapy resulted in earlier pip times (small hole created 12-24 h prior to hatch), and increased hatchling size and weight in the 200 ppt dose groups. However, there appears to be an LED-oil interaction within the oil-treated controls that results in longer hatch times and decreased liver weight within the LED control dose groups in comparison to the non-LED control dose groups. CONCLUSION: Size and hatching times suggest that the hatching success and preparedness of chicks developmentally exposed to dioxin concentrations above the lethality threshold is improved by 670-nm LED treatment administered throughout the gestation period, but the relationship may be complicated by an LED-oil interaction.

Clinical and experimental applications of NIR-LED photobiomodulation.

This review presents current research on the use of far-red to near-infrared (NIR) light treatment in various in vitro and in vivo models. Low-intensity light therapy, commonly referred to as "photobiomodulation," uses light in the far-red to near-infrared region of the spectrum (630-1000 nm) and modulates numerous cellular functions. Positive effects of NIR-light-emitting diode (LED) light treatment include acceleration of wound healing, improved recovery from ischemic injury of the heart, and attenuated degeneration of injured optic nerves by improving mitochondrial energy metabolism and production. Various in vitro and in vivo models of mitochondrial dysfunction were treated with a variety of wavelengths of NIR-LED light. These studies were performed to determine the effect of NIR-LED light treatment on physiologic and pathologic processes. NIRLED light treatment stimulates the photoacceptor cytochrome c oxidase, resulting in increased energy metabolism and production. NIR-LED light treatment accelerates wound healing in ischemic rat and murine diabetic wound healing models, attenuates the retinotoxic effects of methanol-derived formic acid in rat models, and attenuates the developmental toxicity of dioxin in chicken embryos. Furthermore, NIR-LED light treatment prevents the development of oral mucositis in pediatric bone marrow transplant patients. The experimental results demonstrate that NIR-LED light treatment stimulates mitochondrial oxidative metabolism in vitro, and accelerates cell and tissue repair in vivo. NIR-LED light represents a novel, noninvasive, therapeutic intervention for the treatment of numerous diseases linked to mitochondrial dysfunction.

Effects of 670-nm phototherapy on development.

OBJECTIVE: The objective of the present study was to assess the survival and hatching success of chickens (Gallus gallus) exposed in ovo to far-red (670-nm) LED therapy. BACKGROUND DATA: Photobiomodulation by light in the red to near-infrared range (630-1000 nm) using low-energy lasers or light-emitting diode (LED) arrays has been shown to accelerate wound healing and improve recovery from ischemic injury. The mechanism of photobiomodulation at the cellular level has been ascribed to the activation of mitochondrial respiratory chain components resulting in initiation of a signaling cascade that promotes cellular proliferation and cytoprotecton. MATERIALS AND METHODS: Fertile chicken eggs were treated once per day from embryonic days 0-20 with 670-nm LED light at a fluence of 4 J/cm2. In ovo survival and death were monitored by daily candling (after Day 4). RESULTS: We observed a substantial decrease in overall and third-week mortality rates in the light-treated chickens. Overall, there was approximately a 41.5% decrease in mortality rate in the light-treated chickens (NL: 20%; L: 11.8%). During the third week of development, there was a 68.8% decrease in the mortality rate in light-treated chickens (NL: 20%; L: 6.25%). In addition, body weight, crown-rump length, and liver weight increased as a result of the 670-nm phototherapy. Light-treated chickens pipped (broke shell) earlier and had a shorter duration between pip and hatch. CONCLUSION: These results indicate that 670-nm phototherapy by itself does not adversely affect developing embryos and may improve the hatching survival rate.

Repression of hepatobiliary transporters and differential regulation of classic and alternative bile acid pathways in mice during pregnancy.

During pregnancy, proper hepatobiliary transport and bile acid synthesis protect the liver from cholestatic injury and regulate the maternal and fetal exposure to bile acids, drugs, and environmental chemicals. The objective of this study was to determine the temporal messenger RNA (mRNA) and protein profiles of uptake and efflux transporters as well as bile acid synthetic and conjugating enzymes in livers from virgin and pregnant mice on gestational days (GD) 7, 11, 14, and 17 and postnatal days (PND) 1, 15, and 30. Compared with virgins, the mRNAs of most transporters were reduced approximately 50% in pregnant dams between GD11 and 17. Western blot and immunofluorescence staining confirmed the downregulation of Mrp3, 6, Bsep, and Ntcp proteins. One day after parturition, the mRNAs of many uptake and efflux hepatobiliary transporters remained low in pregnant mice. By PND30, the mRNAs of all transporters returned to virgin levels. mRNAs of the bile acid synthetic enzymes in the classic pathway, Cyp7a1 and 8b1, increased in pregnant mice, whereas mRNA and protein expression of enzymes in the alternative pathway of bile acid synthesis (Cyp27a1 and 39a1) and conjugating enzymes (Bal and Baat) decreased. Profiles of transporter and bile acid metabolism genes likely result from coordinated downregulation of transcription factor mRNA (CAR, LXR, PXR, PPARα, FXR) in pregnant mice on GD14 and 17. In conclusion, pregnancy caused a global downregulation of most hepatic transporters, which began as early as GD7 for some genes and was maximal by GD14 and 17, and was inversely related to increasing concentrations of circulating 17ß-estradiol and progesterone as pregnancy progressed.

Increased Nrf2 activation in livers from Keap1-knockdown mice increases expression of cytoprotective genes that detoxify electrophiles more than those that detoxify reactive oxygen species.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor critical for protection against electrophilic and oxidative stress. In a recently engineered mouse with knockdown of kelch-like ECH associated protein 1 (Keap1-kd mice), the cytosolic repressor of Nrf2, there is a 55% decrease in Keap1 mRNA and a 200% increase in Nrf2 protein in liver. Experiments with Nrf2-null mice have demonstrated the effects of a lack of Nrf2. However, little is known about the biological effects of more Nrf2 activation. Accordingly, the hepatic phenotype of Keap1-kd mice, as well as the hepatic mRNA expression of cytoprotective genes were compared among wild-type, Nrf2-null, and Keap1-kd mice. Three distinct patterns of hepatic gene expression were identified among wild-type, Nrf2-null, and Keap1-kd mice. The first pattern encompassed genes that were lower in Nrf2-null mice and considerably higher in Keap1-kd mice than wild-type mice, which included genes mainly responsible for the detoxification and elimination of electrophiles, such as NAD(P)H:quinone oxidoreductase 1 and glutathione-S-transferases (Gst), and multidrug resistance-associated proteins. The second pattern encompassed genes that were lower in Nrf2-null mice but not increased in Keap1-kd mice, and included genes, such as epoxide hydrolase-1, UDP-glucuronosyltransferases, aldehyde dehydrogenases, as well as genes important in the detoxification of reactive oxygen species, such as superoxide dismutase 1 and 2, catalase, and peroxiredoxin 1. The third pattern encompassed genes that were not different among wild-type, Nrf2-null, and Keap1-kd mice and included genes such as glutathione peroxidase, microsomal Gsts, and uptake transporters. In conclusion, the present study suggests that increased activation of hepatic Nrf2 is more important for the detoxification and elimination of electrophiles than reactive oxygen species.

Nrf2 activation enhances biliary excretion of sulfobromophthalein by inducing glutathione-S-transferase activity.

Sulfobromophthalein (BSP) is used to study hepatobiliary excretory function. BSP is conjugated with glutathione (GSH), whereas its dibrominated analog disulfobromophthalein (DBSP) is not conjugated with GSH prior to biliary excretion. In addition, both BSP and DBSP are transported into hepatocytes via organic anion-transporting polypeptides and excreted into bile via multidrug resistance-associated protein 2 (Mrp2). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that under basal conditions is targeted for proteasomal degradation in the cytosol by kelch-like ECH-associated protein 1 (Keap1). Electrophilic and oxidative stress facilitate Nrf2 nuclear translocation and subsequent induction of cytoprotective genes, including GSH synthetic enzymes, GSH-S-transferases (Gsts), and Mrp transporters. The current study determined whether varying the amount of Nrf2 activation would effect the elimination of BSP and DBSP. Male wild-type (WT), Nrf2-null, and Keap1-knockdown (Keap1-kd) mice were administered BSP or DBSP. Within 30 min, Nrf2-null mice excreted 25%, WT mice 52%, and Keap1-kd mice 80% of the injected BSP. Liver GSH content was not altered by BSP. The biliary excretion of GSH and messenger RNA (mRNA) expression of major Gsts were directly proportional to the amount of Nrf2. Moreover, BSP-GSH conjugation activity in the liver of Nrf2-null and Keap1-kd mice was 42% and 237% of WT mice, respectively. In contrast to BSP, there were no differences in biliary excretion or plasma disappearance of DBSP among the three genotypes, suggesting that the modest differences in Mrp2 mRNA expression among genotypes do not affect BSP or DBSP biliary excretion. Collectively, these results indicate that increased biliary excretion of BSP, and possibly other compounds, is due to Nrf2-induced Gst mRNA expression and enzyme activity.

Introducing the "TCDD-inducible AhR-Nrf2 gene battery".

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces genes via the transcription factor aryl hydrocarbon receptor (AhR), including Cyp1a1, NAD(P)H:quinone oxidoreductase 1 (Nqo1), UDP-glucuronosyltransferase 1a6 (Ugt1a6), and glutathione S-transferase a1 (Gsta1). These genes are referred to as the "AhR gene battery." However, Nqo1 is also considered a prototypical target gene of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). In mice, TCDD induction of Nrf2 and Nrf2 target, Nqo1, is dependent on AhR, and thus TCDD induction of drug-processing genes may be routed through an AhR-Nrf2 sequence. There has been speculation that Nrf2 may be involved in the TCDD induction of drug-processing genes; however, the data are not definitive. Therefore, to address whether TCDD induction of Nqo1, Ugts, and Gsts is dependent on Nrf2, we conducted the definitive experiment by administering TCDD (50 mug/kg, ip) to Nrf2-null and wild-type (WT) mice and collecting livers 24 h later to quantify the mRNA of drug-processing genes. TCDD induction of Cyp1a1 and Ugt1a1 was similar in WT and Nrf2-null mice, whereas TCDD induction of Ugt1a5 and 1a9 was blunted in Nrf2-null mice. TCDD induced Nqo1, Ugt1a6, 2b34, 2b35, 2b36, UDP-glucuronic acid-synthesizing gene UDP-glucose dehydrogenase, and Gsta1, m1, m2, m3, m6, p2, t2, and microsomal Gst1 in WT mice but not in Nrf2-null mice. Therefore, the present study demonstrates the novel finding that Nrf2 is required for TCDD induction of classical AhR battery genes Nqo1, Ugt1a6, and Gsta1, as well as most Ugt and Gst isoforms in livers of mice.

Ontogenic expression of hepatic Ahr mRNA is associated with histone H3K4 di-methylation during mouse liver development.

The aryl hydrocarbon receptor (Ahr) is a xenobiotic sensor that regulates the expression of a battery of drug-metabolizing genes. However, Ahr is also important for normal liver development. The purpose of the present study was to examine the ontogeny of Ahr mRNA in mouse liver, and determine the epigenetic mechanisms regulating Ahr gene transcription during postnatal liver development. There was a 224% increase in hepatic Ahr mRNA from 2 days before birth to 45 days after birth. ChIP-on-chip analysis demonstrated that DNA methylation and histone H3K27 tri-methylation (H3K27Me3), two epigenetic marks for suppression of gene transcription, were consistently low around the Ahr gene locus. In contrast, enrichment of histone H3K4 di-methylation (H3K4Me2), a hallmark for gene activation, increased 182% from prenatal to young adult period around the Ahr gene locus. Regression analysis revealed a strong correlation between enrichment of H3K4Me2 and Ahr mRNA (r=0.91). In conclusion, postnatal H3K4Me2 enrichment positively associates with Ahr mRNA in developing mouse liver, providing a permissive chromatin state allowing Ahr gene transactivation in postnatal liver development.

670 nanometer light treatment attenuates dioxin toxicity in the developing chick embryo.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an acutely toxic anthropogenic chemical. Treatment with a red to near-infrared (630-1000 nm) light-emitting diode (LED) attenuates the toxicant-induced oxidative stress and energy deficit in neuronal cell culture. For this study, fertile chicken (Gallus gallus) eggs were injected once at the start of incubation with sunflower oil vehicle or 200 pg TCDD/g egg (200 parts per trillion), an environmentally relevant dose. Daily LED treatment after TCDD exposure reduced embryonic mortality by 47%. LED treatment of TCDD-exposed eggs also decreased the hepatic oxidized-to-reduced glutathione ratio by 88%. Activities of other hepatic indicators of oxidative stress, such as glutathione reductase and catalase, were increased after LED treatment of TCDD-exposed eggs. Our study demonstrates that 670 nm phototherapy can mitigate the oxidative stress and energy deficit resulting from developmental exposure to TCDD while reducing TCDD-induced embryo mortality. Moreover, LED treatment restores hepatic enzyme activities to control levels in TCDD-exposed embryos. The effective attenuation of TCDD-induced embryo toxicity by LED treatment could extend to mitigating the effects of other teratogens that induce oxidative and energy stress.