Altered thyroxin and retinoid metabolic response to 2,3,7,8-tetrachlorodibenzo-p-dioxin in aryl hydrocarbon receptor-null mice

To determine whether the disruption of thyroid hormone and retinoid homeostasis that occurs after exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can be mediated by the arylhydrocarbon receptor (AhR), pregnant AhR-heterozygous (AhR+/–) mice were administered a single oral dose of 10 g kg^–1 TCDD at gestation day 12.5. Serum and liver were collected on postnatal day 21 from vehicle-treated control or TCDD-treated AhR+/– and AhR-null (AhR–/–) mouse pups. Whereas TCDD exposure resulted in a marked reduction of total thyroxin (TT4) and free T4 (FT4) levels in the serum of AhR+/– mice, TCDD had no effects on AhR–/– mice. Gene expression of UDP-glucuronosyltransferase (UGT)1A6, cytochrome P450 (CYP)1A1, and CYP1A2 in the liver was induced markedly by TCDD in AhR+/– but not AhR–/– mice. Induction of CYP1A1 in response to TCDD was confirmed by immunohistochemical evidence in that CYP1A1 protein was conspicuously localized in the cytoplasm of hepatocytes in the centrilobular region. Levels of retinyl palmitate were greatly reduced in the liver of TCDD-exposed AhR+/– mice, but not in vehicle-treated AhR+/– mice. No effects of TCDD on retinoid levels in the liver were found in AhR–/– mice. We conclude that disruption of thyroid hormone and retinoid homeostasis is mediated entirely via AhR. Induction of UGT1A6 is thought to be responsible at least partly for reduced serum thyroid hormone levels in TCDD-exposed mice.     

Antiteratogenic effects of alpha-naphthoflavone on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposed mice in utero

The effects of α-naphthoflavone, an aryl hydrocarbon receptor (AhR) antagonist, on the reproductive toxicity and teratogenicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were investigated. Pregnant C57BL/6J mice were orally administered α-naphthoflavone either once on gestational day 12 (GD12; 50 μg/kg) or for 6 days (GD8–GD13; 5 mg/kg/day) followed by an oral challenge with TCDD (14 μg/kg) on GD12. Cesarean section was performed on GD18 for the evaluation of maternal and fetal toxicities. TCDD caused severe fetal malformations including cleft palate (43.7%) and renal pelvic and ureteric dilatations (100%). The administration of α-naphthoflavone either in a single treatment or 6-days remarkably reduced the incidence of cleft palate to 27.6% and 26.5%, respectively. In addition, the degree of renal pelvic and ureteric dilatations caused by TCDD were significantly attenuated by repeated treatment of α-naphthoflavone. These results suggest that AhR antagonists such as α-naphthoflavone could be promising candidates for reducing the incidence and severity of fetal malformations caused by TCDD exposure in utero.     

Cyp1a1(-/-) male mice: protection against high-dose TCDD-induced lethality and wasting syndrome, and resistance to intrahepatocyte lipid accumulation and uroporphyria

To study liver toxicity and uroporphyrin (URO) accumulation and urinary excretion, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent ligand for the aryl hydrocarbon receptor (AHR), is often used as the prototype. In this study, we asked the question how important is the role of CYP1A1 in causing TCDD toxicity. Using a single large intraperitoneal dose of TCDD (200 microg/kg) and following the response over an 8-week period, we found this dose: (a) was lethal in less than 4 weeks to Cyp1a1(+/+) males but not to Cyp1a1(-/-) males or to females of either genotype; (b) caused a wasting syndrome in Cyp1a1(+/+) but not Cyp1a1(-/-) mice; (c) resulted in thymic atrophy, regardless of gender or genotype; (d) decreased spleen size and caused leukocytopenia in males but not females of either genotype; (e) caused hepatocyte hypertrophy in Cyp1a1(+/+) more so than in Cyp1a1(-/-) mice; (f) increased intrahepatocyte lipids and total liver fat content in Cyp1a1(+/+) more than Cyp1a1(-/-) males and females; and (g) caused uroporphyria in Cyp1a1(+/+) males much more than Cyp1a1(+/+) females, or in Cyp1a1(-/-) mice. Contrary to Cyp1a2(-/-) knockout mice that exhibited 15 times less accumulation of TCDD in liver than Cyp1a1/1a2(+/+) wild-type mice, Cyp1a1(-/-) mice did not show this altered TCDD distribution-indicating that CYP1A2 but not CYP1A1 is the major hepatic TCDD-binding "sink". Our data demonstrate that CYP1A1 contributes to high-dose TCDD-induced toxicity, uroporphyria, and lethality.     

Dose-response relationship of cytochrome P4501b1 mRNA induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin in livers of C57BL/6J and DBA/2J mice

 The dose-response relationship of cytochrome P4501b1 (Cyp1b1) and Cyp1a1 induction in livers of TCDD-treated female C57BL/6J and DBA/2J mice are described. The animals were treated i.p. with 0.001, 0.01, 0.1, 1, 10 and 50 μg TCDD/kg for 24 h, and Cyp1b1 and Cyp1a1 mRNA expression was analyzed by RT-PCR. In the livers of both mouse strains, the Cyp1b1 and Cyp1a1 mRNA content was increased after TCDD exposure in a dose-dependent manner. These effects were more pronounced in TCDD-responsive C57BL/6J mice than in the less responsive DBA/2J mice, although Cyp1a1 was more responsive to TCDD than Cyp1b1 in both strains. The calculated ED₅₀ values for Cyp1b1 and Cyp1a1 induction in livers of TCDD-treated C57BL/6J mice were 1.3 and 0.08 μg TCDD/kg, respectively. The corresponding values for half-maximal induction response in livers of DBA/2J mice were 3.4 μg TCDD/kg for Cyp1b1 and 1.5 μg TCDD/kg for Cyp1a1. These results show that Cyp1b1 mRNA expression is less inducible by TCDD than Cyp1a1. Both genes are highly inducible in TCDD-responsive C57BL/6J mice expressing the high affinity arylhydrocarbon receptor (Ah receptor), suggesting that Cyp1b1, like Cyp1a1, is a potential Ah receptor-regulated gene. Received: 8 December 1995/Accepted: 6 February 1996     

Aryl hydrocarbon receptor‐mediated effects of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin on glucose‐stimulated insulin secretion in mice

Epidemiological and laboratory studies suggested that exposure to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) affects glucose homeostasis and increases the incidence of type 2 diabetes mellitus. To evaluate the effects of TCDD on insulin secretion from islets of Langerhans (islets), we designed in vivo, ex vivo and in vitro experiments. For the in vivo experiment, male C57BL/6J and aryl hydrocarbon receptor (AhR)‐null mice were injected intraperitoneally with TCDD (10 μg kg^?1 b.w.), fasted for 12 h and administered glucose 24 h post‐administration. TCDD exposure significantly decreased the plasma insulin concentration at 60 and 120 min after a glucose challenge in C57BL/6J mice but not in AhR‐null mice. In contrast, the plasma glucose concentration was not changed by TCDD exposure in both C57BL/6J and AhR‐null mice. For the ex vivo experiment, we isolated islets 24 h after TCDD administration and determined the glucose‐stimulated insulin secretion from the islets. The insulin secretion level was found to be significantly decreased by TCDD exposure at 60 min after glucose treatment. For the in vitro experiment, islets harvested from untreated C57BL/6J mice were exposed to 0.1, 1, 10 or 100 nM TCDD for 24 h and analyzed for glucose‐stimulated insulin secretion. Insulin secretion from the islets remained unchanged regardless of TCDD dose. In conclusion, TCDD exposure impaired the second phase of glucose‐stimulated secretion of insulin from the islets via the AhR signaling pathway. Copyright © 2009 John Wiley & Sons, Ltd.     

Modulation of aryl hydrocarbon receptor‐regulated genes by acute administration of ammonium metavanadate in kidney,lung and heart of C57BL/6 mice

We recently reported that vanadium (V⁵⁺) was able to decrease the 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD)‐mediated induction of Cyp1a1 and Nqo1 at mRNA, protein and catalytic activity levels in mouse hepatoma Hepa 1c1c7 and human hepatoma HepG2 cells. However, little is known regarding the in vivo effects. Thus, the objective of this study was to investigate whether similar effects would occur at the in vivo level. Therefore, we examined the effect of exposure to V⁵⁺ (5 mg kg^?1) with or without TCDD (15 µg kg^?1) on the AhR‐regulated genes in kidney, lung and heart of C57BL/6 J mice. Our results demonstrated that V⁵⁺ alone significantly decreased Cyp1b1 protein and catalytic activity levels in kidney at 24 h. Moreover, it significantly potentiated Nqo1 and Gsta1 gene expression in the heart, and only Gsta1 gene expression in the lung. Upon co‐exposure, we found that V⁵⁺significantly inhibited the TCDD‐mediated induction of Cyp1a1, Cyp1a2 and Cyp1b1 mRNA, protein and catalytic activity levels in the kidney at 24 h. On the other hand, V⁵⁺ significantly potentiated the TCDD‐mediated induction of Nqo1 and Gsta1 protein and activity levels in the kidney. Cyp1a1, Cyp1b1, Nqo1 mRNA, protein and catalytic activity levels in the lung were significantly potentiated at 6 h. Interestingly, all tested genes in the heart were significantly decreased at 6 h with the exception of Gsta1 mRNA. The present study demonstrates that V⁵⁺ modulates TCDD‐induced AhR‐regulated genes. Furthermore, the effect on one of these enzymes could not be generalized to other enzymes even if it was in the same organ. Copyright © 2012 John Wiley & Sons, Ltd.     

Inhibitory Effects of Vitamin A on TCDD-induced Cytochrome P-450 1A1 Enzyme Activity and Expression

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an extremely potentenvironmental contaminant that produces a wide range of adversebiological effects, including the induction of cytochrome P4501A1(CYP1A1) that may enhance the toxic effects of TCDD. Severalstudies indicated that concurrent supplementation of vitaminA could reduce the toxicity, and potentially inhibit CYP1A1activity (measured as ethoxyresorufin-O-deethylase [EROD] activity).In the present study, we investigated the in vivo effects ofvitamin A on EROD activities and the expression of CYP1A1 inthe liver of TCDD-treated mice. In Experiment I, the mice weregiven a single oral dose of 40 µg TCDD/kg body weightwith or without the continuous administration of 2500 IU vitaminA/kg body weight/day, and were killed on day 1, 3, 7, 14, or28. In Experiment II, the mice were given daily an oral doseof 0.1 µg TCDD/kg body weight with or without supplementof 2000 IU vitamin A/kg body weight, and were killed on day14, 28, or 42. In both experiments, TCDD caused liver damageand increase in relative liver weights, augmented the EROD activitiesand CYP1A1 expression, and increased the aryl hydrocarbon receptor(AhR) mRNA expression, but did not alter the AhR nuclear translocator(ARNT) mRNA expression. CYP1A1 mRNA expression and AhR mRNAexpression showed a similar time course. The liver damage inTCDD + vitamin A–treated mice was less severe than thatin TCDD-treated mice. EROD activities, CYP1A1 expression, andAhR mRNA expression in vitamin A + TCDD–treated mice werelower than those in TCDD-treated mice, indicating that supplementationof vitamin A might attenuate the liver damage caused by TCDD.     

Effect of mercury on aryl hydrocarbon receptor-regulated genes in the extrahepatic tissues of C57BL/6 mice

The individual toxic effects of aryl hydrocarbon receptors (AhR) ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or heavy metals typified by mercury (Hg²⁺) has been previously demonstrated. However, little is known about the combined toxic effects of TCDD and Hg²⁺in vivo. Therefore, we examined the effect of exposure to Hg²⁺ (2.5 mg/kg) in the absence and presence of TCDD (15 μg/kg) on the AhR-regulated genes using C57Bl/6 mice. Hg²⁺ alone did not affect kidney, lung, or heart Cyp1a1/1a2/1b1 mRNA levels. On the contrary, Hg²⁺ alone significantly induced kidney Cyp1a1/1a2/1b1 and lung Cyp1b1 protein and catalytic activities. Hg²⁺ also induced Nqo1, Gsta1, and HO-1 at the mRNA, protein, and activity levels in the kidney and heart but not in the lung. Upon co-exposure to Hg²⁺ and TCDD, Hg²⁺ significantly potentiated the TCDD-mediated induction of kidney and lung Cyp1a1/1a2/1b1 mRNA levels, while it decreased their kidney protein and catalytic activity and it increased their lung protein. In addition, Hg²⁺ potentiated the TCDD-mediated induction of Nqo1, Gsta1, and HO-1 at mRNA, protein and activity levels in all tissues. The present study demonstrates that Hg²⁺ modulates the constitutive and TCDD-induced AhR-regulated genes in a time-, tissue- and, AhR-regulated enzyme genes manner.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) blocks ovulation by a direct action on the ovary without alteration of ovarian steroidogenesis: lack of a direct effect on ovarian granulosa and thecal-interstitial cell steroidogenesis in vitro

The main purpose of this study was to investigate the direct effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on ovarian function including ovulation and steroidogenesis. In vivo effects of TCDD were investigated on ovulation and alteration of circulating and ovarian steroid hormones in immature hypophysectomized rats (IHR) primed with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). In addition, in vitro effects of TCDD on the steroidogenesis of granulosa cells (GC), theca-interstitial cells (TIC), and whole ovarian dispersates derived from the ovary of IHR were investigated. In the ovulation model, rats were hypophysectomized on Day 23 of age. On Day 26, the IHR were given 20 μg TCDD/kg by gavage. The next day eCG (10 IU) was injected sc to stimulate follicular development. Fifty-two hours after eCG, 10 IU hCG was given to induce ovulation. TCDD (20 μg/kg) blocked ovulation and reduced ovarian weight in IHR. Concentrations of progesterone (P4), androstenedione (A4), and estradiol (E2) in sera and ovaries were not altered by TCDD at 12, 24, 48, and 72 h after eCG, except for a two-fold increase in ovarian concentration of A4 at 48 h after TCDD. However, this higher concentration of A4 at 48 h after TCDD did not reflect that of A4 in sera and did not correlate with E2 in either sera or ovaries. In isolated GC from untreated IHR, TCDD (0.1 to 100 nM) had no significant effect on P4 and E2 after stimulation by LH or FSH. In TIC and whole ovarian dispersates containing GC, TIC, and other ovarian cells, TCDD (0.1 to 800 nM) had no effect on A4 and P4 secretion stimulated by LH. Using RT-PCR, AhR mRNA was shown to be expressed constitutively in the whole ovary of IHR with maximum down-regulation at 6 h after TCDD (20 μg/kg). Ovarian CYP1A1 was induced maximally at 6 h after TCDD, whereas CYP1B1 could not be detected. The induction of AhR related genes by TCDD in the ovary implies the existence of AhR-mediated signal transduction pathways. In summary, these results indicate that TCDD does not affect ovulation in IHR by altering ovarian steroidogenesis. It seems that inhibition of ovulation by TCDD is due to processes related to follicular rupture.     

Protection of the Cyp1a2(-/-) null mouse against uroporphyria and hepatic injury following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the liver of C57BL/6J mice is a model for clinical sporadic porphyria cutanea tarda (PCT). There is massive uroporphyria, inhibition of uroporphyrinogen decarboxylase (UROD) activity, and hepatocellular damage. A variety of evidence implicates the CYP1A2 enzyme as necessary for mouse uroporphyria. Here we report that, 5 weeks after a single oral dose of TCDD (75 microg/kg), Cyp1a2(+/+) wild-type mice showed severe uroporphyria and greater than 90% decreases in UROD activity; in contrast, despite exposure to this potent agent Cyp1a2(-/-) knockout mice displayed absolutely no increases in hepatic porphyrin levels, even after prior iron overload, and no detectable inhibition of UROD activity. Plasma levels of alanine-aminotransferase (ALT) and aspartate aminotransferase (AST)-although elevated in both genotypes after TCDD exposure-were significantly less in Cyp1a2(-/-) than in Cyp1a2(+/+) mice, suggesting that the absence of CYP1A2 also affords partial protection against TCDD-induced liver toxicity. Histological examination confirmed a decrease in hepatocellular damage in TCDD-treated Cyp1a2(-/-) mice; in particular, there was no bile duct damage or proliferation that in the Cyp1a2(+/+) mice might be caused by uroporphyrin. We conclude that CYP1A2 is both necessary and essential for the potent uroporphyrinogenic effects of TCDD in mice, and that CYP1A2 also plays a role in contributing to TCDD-induced hepatocellular injury. This study has implications for both the toxicity assessment of TCDD and the hepatic injury seen in PCT patients.     

Fibroblast growth factor (Fgf) 21 is a novel target gene of the aryl hydrocarbon receptor (AhR)

The toxic effects of dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), mainly through activation of the aryl hydrocarbon receptor (AhR) are well documented. Fibroblast growth factor (Fgf) 21 plays critical roles in metabolic adaptation to fasting by increasing lipid oxidation and ketogenesis in the liver. The present study was performed to determine whether activation of the AhR induces Fgf21 expression. In mouse liver, TCDD increased Fgf21 mRNA in both dose- and time-dependent manners. In addition, TCDD markedly increased Fgf21 mRNA expression in cultured mouse and human hepatocytes. Moreover, TCDD increased mRNA (in liver) and protein levels (in both liver and serum) of Fgf21 in wild-type mice, but not in AhR-null mice. Chromatin immunoprecipitation assays showed that TCDD increased AhR protein binding to the Fgf21 promoter (− 105/+ 1 base pair). Fgf21-null mice administered 200 μg/kg of TCDD died within 20 days, whereas wild-type mice receiving the same treatment were still alive at one month after administration. This indicates that TCDD-induced Fgf21 expression protects against TCDD toxicity. Diethylhexylphthalate (DEHP) pretreatment attenuated TCDD-induced Fgf21 expression in mouse liver and white adipose tissue, which may explain a previous report that DEHP pretreatment decreases TCDD-induced wasting. In conclusion, Fgf21 appears to be a target gene of AhR-signaling pathway in mouse and human liver.     

Modulation of aryl hydrocarbon receptor regulated genes by acute administration of trimethylarsine oxide in the lung,kidney and heart of C57BL/6 mice

Abstract

1.?Arsenite alters the expression of aryl hydrocarbon receptor (AhR)-regulated genes in extrahepatic tissues; yet, the effect of organic arsenicals still unknown. Therefore, C57BL/6 mice received trimethylarsine oxide (TMAO; 13?mg/kg i.p.) with or without 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 15?μg/kg), and euthanized at 6 or 24?h.

2.?Our results demonstrated that TMAO increased Cyp1a1 and Cyp1b1 mRNA, protein and activity in the lung. TMAO potentiated the TCDD-mediated induction of Cyp1a1 and Cyp1a2 mRNA, protein and activity in the lung. In the kidney, TMAO increased Cyp1b1 mRNA and protein. TMAO potentiated the TCDD-mediated induction of Cyp1a1 and Cyp1b1 mRNA, protein and activity. In the heart, TMAO potentiated the TCDD-mediated induction of Cyp1a1 and Cyp1b1 mRNA.

3.?Moreover, TMAO induced Nqo1 mRNA in the lung, kidney and heart, with subsequent increase in Nqo1 protein and activity in the lung. TMAO increased Gsta mRNA in the heart; and increased Gsta protein and activity in the lung and kidney. TMAO increased Nqo1 mRNA as compared to TCDD in the kidney and heart, and potentiated the TCDD-mediated induction of Gsta protein and activity in the kidney.

4.?In conclusion, TMAO modulates AhR-regulated genes in a tissue- and enzyme-specific manner.     

Aryl hydrocarbon receptor-mediated induction of Stearoyl-CoA desaturase 1 alters hepatic fatty acid composition in TCDD-elicited steatosis

2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD) induces hepatic dyslipidemia mediated by the aryl hydrocarbon receptor (AhR). Stearoyl-CoA desaturase 1 (Scd1) performs the rate-limiting step in monounsaturated fatty acid (MUFA) synthesis, desaturating 16:0 and 18:0 into 16:1n7 and 18:1n9, respectively. To further examine the role of Scd1 in TCDD-induced hepatotoxicity, comparative studies were performed in Scd1(+/+) and Scd1(-/-) mice treated with 30 μg/kg TCDD. TCDD induced Scd1 activity, protein, and messenger RNA (mRNA) levels approximately twofold. In Scd1(+/+) mice, hepatic effects were marked by increased vacuolization and inflammation and a 3.5-fold increase in serum alanine aminotransferase (ALT) levels. Hepatic triglycerides (TRGs) were induced 3.9-fold and lipid profiling by gas chromatography-mass spectroscopy measured a 1.9-fold increase in fatty acid (FA) levels, consistent with the induction of lipid transport genes. Induction of Scd1 altered FA composition by decreasing saturated fatty acid (SFA) molar ratios 8% and increasing MUFA molar ratios 9%. Furthermore, ChIP-chip analysis revealed AhR enrichment (up to 5.7-fold), and computational analysis identified 16 putative functional dioxin response elements (DREs) within Scd1 genomic loci. Band shift assays confirmed AhR binding with select DREs. In Scd1(-/-) mice, TCDD induced minimal hepatic vacuolization and inflammation, while serum ALT levels remained unchanged. Although Scd1 deficiency attenuated TCDD-induced TRG accumulation, overall FA levels remained unchanged compared with Scd1(+/+) mice. In Scd1(-/-) mice, TCDD induced SFA ratios 8%, reduced MUFA ratios 13%, and induced polyunsaturated fatty acid ratios 5% relative to treated Scd1(+/+) mice. Collectively, these results suggest that AhR regulation of Scd1 not only alters lipid composition but also contributes to the hepatotoxicity of TCDD.     

Urinary excretion kinetics of p-nitrophenol following oral administration of parathion in the rabbit

The urinary excretion kinetics of p-nitrophenol were studied in rabbits following oral administration of parathion at a dose of 3 mg/kg. Elimination of p-nitrophenol began rapidly, and of the total amount excreted during the study period, 46% was excreted in the first 3 h; 85% was excreted at 6 h after administration of the pesticide. The mean maximum excretion rate of p-nitrophenol was 111.15±61.02 g/h reached in a time of 0.77±0.26 h. The formation and disappearance rate constants of the metabolite were 2.85±2.80 h^–1 and 0.80±0.28 h^–1, respectively. A linear relationship was observed between the plasma concentrations of parathion and the urinary excretion rate of p-nitrophenol.     

Morphine Antinociception Is Enhanced in mdr1a Gene-Deficient Mice

Purpose. Previous studies have suggested that P-glycoprotein (P-gp)modulates opioid antinociception for selected -and -agonists. Thisstudy was undertaken to assess morphine antinociception in micelacking the mdr1a gene for expression of P-gp in the CNS. Methods. Morphine (n = 4–5/group) was administered as a single s.c.dose to mdr1a(–/–) mice (3–5 mg/kg) or wild-type FVB controls(8–10 mg/kg). Tail-flick response to radiant heat, expressed as percentof maximum response (%MPR), was used to determine theantinociceptive effect of morphine. Concentrations in serum, brain tissue, andspinal cord samples obtained immediately after the tail-flick test weredetermined by HPLC with fluorescence detection. Parallel experimentswith R(+)-verapamil, a chemical inhibitor of P-gp, also were performedto further investigate the effect of P-gp on morphine-associatedantinociception. Results. Morphine-associated antinociception was increasedsignificantly in the mdr1a(–/–) mice. The ED₅₀ for morphine was > 2-foldlower in mdr1a(–/–) (3.8 ± 0.2 mg/kg) compared to FVB (8.8 ±0.2 mg/kg) mice. However, the EC;i5;i0 derived from the brain tissuewas similar between the two mouse strains (295 ng/g vs. 371 ng/g).Pretreatment with R(+)-verapamil produced changes similar to thoseobserved in gene-deficient mice. P-gp does not appear to affectmorphine distribution between spinal cord and blood, as the spinalcord:serum morphine concentration ratio was similar betweengene-deficient and wild-type mice (0.47 ± 0.03 vs. 0.56 ± 0.04, p > 0.05). Conclusions. The results of this study are consistent with thehypothesis that P-gp attenuates the antinociceptive action of morphine bylimiting the brain:blood partitioning of the opioid.