Across-model spread and shrinking in predicting peatland carbon dynamics under global change.

Large across-model spread in simulating land carbon (C) dynamics has been ubiquitously demonstrated in model intercomparison projects (MIPs), and became a major impediment in advancing climate change prediction. Thus, it is imperative to identify underlying sources of the spread. Here, we used a novel matrix approach to analytically pin down the sources of across-model spread in transient peatland C dynamics in response to a factorial combination of two atmospheric CO2 levels and five temperature levels. We developed a matrix-based MIP by converting the C cycle module of eight land models (i.e., TEM, CENTURY4, DALEC2, TECO, FBDC, CASA, CLM4.5 and ORCHIDEE) into eight matrix models. While the model average of ecosystem C storage was comparable to the measurement, the simulation differed largely among models, mainly due to inter-model difference in baseline C residence time. Models generally overestimated net ecosystem production (NEP), with a large spread that was mainly attributed to inter-model difference in environmental scalar. Based on the sources of spreads identified, we sequentially standardized model parameters to shrink simulated ecosystem C storage and NEP to almost none. Models generally captured the observed negative response of NEP to warming, but differed largely in the magnitude of response, due to differences in baseline C residence time and temperature sensitivity of decomposition. While there was a lack of response of NEP to elevated CO2 (eCO2 ) concentrations in the measurements, simulated NEP responded positively to eCO2 concentrations in most models, due to the positive responses of simulated net primary production. Our study used one case study in Minnesota peatland to demonstrate that the sources of across-model spreads in simulating transient C dynamics can be precisely traced to model structures and parameters, regardless of their complexity, given the protocol that all the matrix models were driven by the same gross primary production and environmental variables.

Photosynthetic responses of Larix kaempferi and Pinus densiflora seedlings are affected by summer extreme heat rather than by extreme precipitation.

The frequency and intensity of summer extreme climate events are increasing over time, and have a substantial negative effect on plants, which may be evident in their impact on photosynthesis. Here, we examined the photosynthetic responses of Larix kaempferi and Pinus densiflora seedlings to extreme heat (+ 3 °C and + 6 °C), drought, and heavy rainfall by conducting an open-field multifactor experiment. Leaf gas exchange in L. kaempferi showed a decreasing trend under increasing temperature, showing a reduction in the stomatal conductance, transpiration rate, and net photosynthetic rate by 135.2%, 102.3%, and 24.8%, respectively, in the + 6 °C treatment compared to those in the control. In contrast, P. densiflora exhibited a peak function in the stomatal conductance and transpiration rate under + 3 °C treatment. Furthermore, both species exhibited increased total chlorophyll contents under extreme heat conditions. However, extreme precipitation had no marked effect on photosynthetic activities, given the overall favorable water availability for plants. These results indicate that while extreme heat generally reduces photosynthesis by triggering stomatal closure under high vapor pressure deficit, plants employ diverse stomatal strategies in response to increasing temperature, which vary among species. Our findings contribute to the understanding of mechanisms underlying the photosynthetic responses of conifer seedlings to summer extreme climate events.

Magnetic quasi-atomic electrons driven reversible structural and magnetic transitions between electride and its hydrides.

In electrides, interstitial anionic electrons (IAEs) in the quantized energy levels at cavities of positively charged lattice framework possess their own magnetic moment and interact with each or surrounding cations, behaving as quasi-atoms and inducing diverse magnetism. Here, we report the reversible structural and magnetic transitions by the substitution of the quasi-atomic IAEs in the ferromagnetic two-dimensional [Gd2C]2+·2e- electride with hydrogens and subsequent dehydrogenation of the canted antiferromagnetic Gd2CHy (y > 2.0). It is demonstrated that structural and magnetic transitions are strongly coupled by the presence or absence of the magnetic quasi-atomic IAEs and non-magnetic hydrogen anions in the interlayer space, which dominate exchange interactions between out-of-plane Gd-Gd atoms. Furthermore, the magnetic quasi-atomic IAEs are inherently conserved by the hydrogen desorption from the P[Formula: see text] 1m structured Gd2CHy, restoring the original ferromagnetic state of the R[Formula: see text]m structured [Gd2C]2+·2e- electride. This variable density of magnetic quasi-atomic IAEs enables the quantum manipulation of floating electron phases on the electride surface.

Pseudo-single domain colloidal superparamagnetic nanoparticles designed at a physiologically tolerable AC magnetic field for clinically safe hyperthermia.

Magnetic nanofluid hyperthermia (MNFH) with pure superparamagnetic nanoparticles (P-SPNPs) has drawn a huge attraction for cancer treatment modality. However, the low intrinsic loss power (ILP) and attributable degraded-biocompatibility resulting from the use of a heavy dose of P-SPNP agents as well as low heat induction efficiency in biologically safe AC magnetic field (HAC,safe) are challenging for clinical applications. Here, we report an innovatively designed pseudo-single domain-SPNP (PSD-SPNP), which has the same translational advantages as that of conventional P-SPNPs but generates significantly enhanced ILP at HAC,safe. According to the analyzed results, the optimized effective relaxation time, τeff, and magnetic out-of-phase susceptibility, χ'', precisely determined by the particle size at the specific frequency of HAC,safe are the main reasons for the significantly enhanced ILP. Additionally, in vivo MNFH studies with colloidal PSD-SPNPs strongly demonstrated that it can be a promising agent for clinically safe MNFH application with high efficacy.

Diazepam metabolism in the kidneys of male and female rats of various strains.

Previously, we have reported drastic strain differences of diazepam metabolism in the livers of a variety of rat strain. In this study, to characterize strain and sex differences of diazepam metabolism in the kidney, renal microsomal diazepam metabolic activities were determined in the Dark Agouti (DA), Sprague-Dawley (SD), Brown Norway (BN) and Wistar (WS) strains of rat. We found that the major pathway of diazepam metabolism in the kidney was diazepam N-demethylation, which is different from that in the liver, 3-hydroxylation. A Dose-course (12.5-200 muM of diazepam) study revealed that the DA and WS male rats had higher diazepam N-demethylation activity than the SD and BN rats. In contrast to the males, a lower activity of diazepam N-demethylation was observed in female BN rats. By Western blot analysis, constitutive protein expressions of cytochrome P450 (CYP) 2C11, which is responsible for diazepam N-demethylation, were detected in the 4 strain in both the male and female rats, and the BN rats had lower expression levels of CYP2C11 protein. However, we did not observe significant differences in the kinetic parameters of diazepam N-demethylation. Our results suggested that there was a strain difference in CYP-dependent diazepam N-demethylation in the rat kidney, which is different from the finding in liver microsomes.

Outcomes of Combined Bone Marrow Stimulation and Patch Augmentation for Massive Rotator Cuff Tears.

BACKGROUND: The high failure rate after surgical repair of massive rotator cuff tears is a consistent problem. PURPOSE: To evaluate the clinical and radiological outcomes of arthroscopic rotator cuff repair with bone marrow stimulation and patch augmentation in patients with massive rotator cuff tears. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: This study included 21 patients who underwent bone marrow stimulation and patch augmentation (group 1) and 54 patients who underwent conventional repair (group 2) for massive rotator cuff tears. Postoperative clinical outcomes were evaluated based on visual analog scale (VAS) for pain, simple shoulder test (SST), University of California, Los Angeles (UCLA), Constant, and American Shoulder and Elbow Surgeons (ASES) scores at baseline, 1 year postoperatively, and final follow-up. Anatomic outcomes were evaluated by using postoperative magnetic resonance imaging at 1 year after surgery. RESULTS: No significant differences in demographic characteristics and baseline data were observed between groups 1 and 2. Clinical symptoms were significantly improved at the final follow-up in both groups (P < .001). At the final follow-up, no significant differences were found in VAS pain (P = .676), SST (P = .598), UCLA (P = .100), Constant (P = .469), or ASES (P = .880) scores. However, the retear rate was lower in group 1 (4/21, 19.0%) than in group 2 (25/54, 46.3%) (P = .036), and the medial-row failure rate (type 2 retears) was much lower in group 1 (0/4, 0%) than in group 2 (18/25, 72.0%) (P = .014). CONCLUSION: Concomitant bone marrow stimulation and patch augmentation significantly reduced retear and medial-row failure rates in the arthroscopic repair of massive rotator cuff tears.

Protective effect of resveratrol against cisplatin-induced ototoxicity in HEI-OC1 auditory cells.

OBJECTIVES: Cisplatin is an effective chemotherapeutic drug, but it generates reactive oxygen species (ROS) that induce severe adverse effects such as ototoxicity. Resveratrol reportedly prevents oxidative stress-induced cell death. Thus, we hypothesized that the anti-oxidative effect of resveratrol could protect against cisplatin-induced ototoxicity. The present study examined the protective effect of resveratrol against cisplatin-induced ototoxicity in HEI-OC1 auditory cells. METHODS: HEI-OC1 cells were pretreated with resveratrol at 1µM for 24h and then exposed to 15µM cisplatin for 48h. Resulting cytotoxicity was measured by the MTT method, and intracellular ROS was measured using flow cytometry. Protective effect of resveratrol was compared with other anti-oxidants. RESULTS: Pretreatment with resveratrol 1µM protected HEI-OC1 auditory cells against cisplatin-induced cytotoxicity and significantly reduced a cisplatin-induced increase in ROS. Resveratrol provided significant protection against 15µM cisplatin applied for 48h (50.8% cell viability in the cisplatin group vs. 57.6% in the cisplatin-plus-resveratrol group), and there was a 9% decrease in cisplatin-induced ROS associated with resveratrol. CONCLUSIONS: This is the study investigating the protective effects of resveratrol against cisplatin-induced ototoxicity in an auditory cell line. Resveratrol significantly reduced a cisplatin-induced increase in ROS and thereby inhibited cisplatin-induced cytotoxicity.

Polymorphism in diazepam metabolism in Wistar rats.

We observed variations in the metabolism of diazepam in Wistar rats. We studied these variations carefully, and found that the variations are dimorphic and about 17% of male rats of Wistar strain we examined showed two times higher diazepam metabolic activities in their liver microsomes than the rest of animals at the substrate concentrations less than 5 microM. We classified them as extensive metabolizer (EM) and poor metabolizer (PM) of diazepam. No sex difference was observed in the frequency of appearance of EM. Activities of the primary metabolic pathways of diazepam were examined to elucidate the cause of this polymorphism in male Wistar rats. No significant differences were observed in activities of neither diazepam 3-hydroxylation or N-desmethylation between EM and PM rats, while activity of diazepam p-hydroxylation was markedly (more than 200 times) higher in EM rats, indicating that this reaction is responsible for the polymorphism of diazepam metabolism in Wistar rats. We examined the expression levels of CYP2D1, which was reported to catalyze diazepam p-hydroxylation in Wistar rats to find no differences in the expression levels of CYP2D1 between EM and PM rats. The kinetic study on diazepam metabolism in male Wistar rats revealed that EM rats had markedly higher V(max) and smaller K(m) in diazepam p-hydroxylation than those of PM rats, indicating the presence of high affinity high capacity p-hydroxylase enzyme in EM rats. As a consequence, at low concentrations of diazepam, major pathways of diazepam metabolism were p-hydroxylation and 3-hydroxylation in male EM rats, while in male PM rats, 3-hydroxylation followed by N-desmethylation. Due to this kinetic nature of p-hydroxylase activity, EM rats had markedly higher total CL(int) of diazepam than that of PM rats. Polymorphism in diazepam metabolism in humans is well documented, but this is the first report revealing the presence of the polymorphism in diazepam metabolism in rats. The current results infer polymorphic expression of new diazepam p-hydroxylating enzyme with lower K(m) than CYP2D1 in EM Wistar rats.

DI-(2-ethylhexyl) phthalate-induced cell proliferation is involved in the inhibition of gap junctional intercellular communication and blockage of apoptosis in mouse Sertoli cells.

Di-(2-ethylhexyl) phthalate (DEHP) has been studied on gap junctional intercellular communication (GJIC) and apoptosis in cultured normal mouse Sertoli cells. Since the inhibition of GJIC and programmed cell death or apoptosis play important roles in tumor promotion and developmental toxicity, it has been hypothesized that tumor promoters may inhibit apoptosis by blocking GJIC. The results showed that the most significant downregulation of GJIC was detected at 9 h after DEHP treatment. However, a significant concentration-dependent pattern was not observed at concentrations of 100 and 500 microM, but there was a time-dependent recovery of GJIC. DEHP inhibited the apoptotic changes in the cells such as chromatin condensation, nuclear fragmentation, and the cleavage of poly(ADP-ribose) polymerase. Morphological changes related to apoptosis appeared in the nontreated cells after 12 h of serum deprivation. These morphological changes were significantly reduced in the TM5 Sertoli cells treated with 500 microM DEHP for 24 h. These results suggest that DEHP inhibited apoptosis in this cell line, preceded by the downregulation of GJIC. It was also found that DEHP reduced the phosphorylation of Cx43, which might partly explain the mechanism of inhibition of GJIC.

Alterations of activities of cytosolic phospholipase A2 and arachidonic acid-metabolizing enzymes in di-(2-ethylhexyl)phthalate-induced testicular atrophy.

Di-(2-ethylhexyl) phthalate (DEHP), a peroxisome proliferator-activated receptor alpha (PPARalpha) ligand, alters the lipid composition of rat testis, yet the mechanism is unclear. In this study, we investigated the effect of DEHP on the synthesis and metabolism of arachidonic acid (AA), a precursor of eicosanoids, in the testis of prepubertal rats. DEHP (100 and 1,000 mg/kg, 5 days) administration caused a significant reduction in activity of cytosolic phospholipase A2 (cPLA2), the rate-limiting enzyme in the AA and eicosanoid synthesis pathways. DEHP increased the expression of 12-lipoxygenase (12-LOX) in rat testis, whereas cyclooxygenase-2 (COX-2) expression was not altered. Cytochrome P450 4A1 (CYP4A1), a product of a PPARalpha-regulated gene, was markedly increased in the testis by DEHP administration. Taken together, DEHP suppresses cPLA2 activity and induces the AA metabolizing enzymes such as 12-LOX and CYP4A1, resulting in the reduction of AA level. These data suggest that altered AA metabolic cascades may be related to the decrease of testosterone concentration in DEHP-induced testicular atrophy.

Nail-preserving excision for subungual glomus tumour of the hand.

Subungual glomus tumours can cause excruciating pain and nail deformity. Conventional surgical excision requires nail removal and, therefore, nail deformity often occurs. Because nail preservation prevents further damage to the nail bed, it is beneficial for patients from the perspectives of pain and cosmesis. Here, the authors introduce a nail-preserving transungual approach for subungual glomus tumours. Between 1996-2010, 34 patients were treated using this nail-preserving transungual approach for the excision of a subungual glomus tumour and were followed up. Preoperatively, all patients complained of pain (mean visual analogue scale (VAS) 8.9), and seven of the 34 patients presented concomitant nail deformities. During surgeries, nails were elevated after incising nail folds, and tumours were excised after a longitudinal nail bed incision. Elevated nails were relocated to their original position after nail bed repair. Thirty-two of the 34 patients achieved complete recovery without sign of recurrence. Mean postoperative pain was reduced (VAS 0.9, range = 0-2), and preoperative nail deformity was also improved. The nail preserving transungual approach provides several advantages, that is, better nail bed exposure, resulting in easier tumour excision, and less damage to the nail bed with less deformity of the nail.

Combined-modality radioimmunotherapy: synergistic effect of paclitaxel and additive effect of bevacizumab.

INTRODUCTION: This study was undertaken to investigate the effect of paclitaxel and bevacizumab on the therapeutic efficacy of (90)Y-labeled B3 monoclonal antibody, directed against Le(y) antigen, for the treatment of Le(y)-positive A431 tumors implanted subcutaneously in the right hind flank of nude mice. METHODS: When the tumor size reached ~200 mm(3), the mice received a single dose of intravenous (iv) (90)Y-labeled B3 (60 µCi/150 µg or 100 µCi/150 µg B3), intraperitoneal paclitaxel (40 mg/kg) or iv bevacizumab (5 mg/kg) for monotherapy. To investigate the effect of combined therapies on survival, the mice were treated with two or three agents in the following combinations: (90)Y-B3 on day 0 and paclitaxel on day 1; bevacizumab on -1 day and (90)Y-B3 on day 0; bevacizumab on -1 day and paclitaxel on day 1; bevacizumab, (90)Y-B3 and paclitaxel each at 1-day intervals. The mice with no treatment were used as a control. The tumor volume at 1000 mm(3) was used as a surrogate end point of survival. RESULTS: Compared to control animals, paclitaxel delayed tumor growth with a significantly longer median survival time (P<.001), whereas bevacizumab alone showed a less pronounced effect on a median survival time (P=.18). (90)Y-B3 increased the median survival time in a dose-dependent manner (P<.05). The combined therapy of bevacizumab with paclitaxel produced a trend toward an increase of the median survival time compared to paclitaxel alone (P=.06), whereas bevacizumab combined with (90)Y-B3 showed a statistically insignificant increase in the median survival time compared to (90)Y-B3 alone (P=.25). The tumor sizes of all animals in these groups reached the surrogate end point of survival by day 35. In contrast, the combined therapy involving (90)Y-B3 with paclitaxel showed a striking synergistic effect in shrinking tumors and prolonging the survival time (P<.001); on day 120, three of nine mice (33%) and six of six mice (100%) were alive without tumor when treated with 60 µCi (90)Y-B3 and 100 µCi (90)Y-B3, respectively. The addition of bevacizumab treatment 1 day before the combined therapy of 60 µCi (90)Y-B3 with paclitaxel did not produce a statistically significant increase in survival when compared to the (90)Y-B3 with paclitaxel (P>.10). Fluorescence microscopy analysis indicated that paclitaxel increased, whereas bevacizumab decreased, the accumulation and penetration of Alexa Fluor 647-B3 into tumor microenvironment compared to the control (P<.05). CONCLUSION: Our findings on the paclitaxel effect support a hypothesis that the increased tumor accumulation and penetration of (90)Y-B3 as well as the high radiosensitization of tumor cells by paclitaxel may be the major factors responsible for the synergistic effect of the combined therapy involving (90)Y-B3 with paclitaxel.

Pulsed high intensity focused ultrasound increases penetration and therapeutic efficacy of monoclonal antibodies in murine xenograft tumors.

The success of radioimmunotherapy for solid tumors remains elusive due to poor biodistribution and insufficient tumor accumulation, in part, due to the unique tumor microenvironment resulting in heterogeneous tumor antibody distribution. Pulsed high intensity focused ultrasound (pulsed-HIFU) has previously been shown to increase the accumulation of (111)In labeled B3 antibody (recognizes Lewis(y) antigen). The objective of this study was to investigate the tumor penetration and therapeutic efficacy of pulsed-HIFU exposures combined with (90)Y labeled B3 mAb in an A431 solid tumor model. The ability of pulsed-HIFU (1 M Hz, spatial averaged temporal peak intensity=2685 W cm(-2); pulse repetition frequency=1 Hz; duty cycle=5%) to improve the tumor penetration and therapeutic efficacy of (90)Y labeled B3 mAb ((90)Y-B3) was evaluated in Le(y)-positive A431 tumors. Antibody penetration from the tumor surface and blood vessel surface was evaluated with fluorescently labeled B3, epi-fluorescent microscopy, and custom image analysis. Tumor size was monitored to determine treatment efficacy, indicated by survival, following various treatments with pulsed-HIFU and/or (90)Y-B3. The pulsed-HIFU exposures did not affect the vascular parameters including microvascular density, vascular size, and vascular architecture; although 1.6-fold more antibody was delivered to the solid tumors when combined with pulsed-HIFU. The distribution and penetration of the antibodies were significantly improved (p-value<0.05) when combined with pulsed-HIFU, only in the tumor periphery. Pretreatment with pulsed-HIFU significantly improved (p-value<0.05) survival over control treatments.

Induction of apoptosis and CYP4A1 expression in Sprague-Dawley rats exposed to low doses of perfluorooctane sulfonate.

In previous studies, perfluorooctane sulfonate (PFOS), an environmental organic compound, was reported to cause hepatotoxicity and hypolipidemia in rodents. However, the low dose toxicity of PFOS and the toxic mechanisms involved remain to be determined. To clarify the low dose toxicity and action mechanism in the target organ toxicity, Sprague-Dawley (SD) rats were orally administered with PFOS at the doses of 0, 1.25, 5, 10 mg/kg/day for 28 days. As a result, no death or abnormal symptoms were observed in all groups. The significant loss of mean body weight was observed in female rats treated with 10 mg/kg PFOS and the relative liver weight of 10 mg/kg PFOS-treated group was significantly greater compared to control. Histopathological examination revealed that fatty change was evident in the liver of male rats treated with PFOS (5 and 10 mg/kg) and hypertrophy and cellular swellings in females at the dose of 10 mg/kg, which showed different pattern of pathological lesions. In addition, we demonstrated the expression induction of hepatic caspase-3 and cytochrome P450 4A1 (CYP4A1) related with apoptosis and lipid metabolism, respectively. This study suggested that no-observed-adverse-effect level (NOAEL) of PFOS was 1.25 mg/kg in 28-day repeated toxicity study and, however, the toxic response showed gender differences. The possible toxic mechanism of PFOS was the induction of apoptosis and altering lipid metabolism which resulted in hepatotoxicity.

Effect of chelator conjugation level and injection dose on tumor and organ uptake of 111In-labeled MORAb-009, an anti-mesothelin antibody.

INTRODUCTION: Radiolabeling of a monoclonal antibody (mAb) with a metallic radionuclide requires the conjugation of a bifunctional chelator to the mAb. The conjugation, however, can alter the physical and immunological properties of the mAb, consequently affecting its tumor-targeting pharmacokinetics. In this study, we investigated the effect of the amount of 2-(p-isothiocyanatobenzyl)-cyclohexyl-diethylenetriamine-pentaacetic acid (CHX-A″) conjugated to MORAb-009, a mAb directed against mesothelin, and the effect of MORAb dose on the biodistribution of (111)In-labeled MORAb-009. METHODS: We used nude mice bearing the A431/K5 tumor as a mesothelin-positive tumor model and the A431 tumor as a mesothelin-negative control. To find the optimal level of CHX-A″ conjugation, CHX-A″-MORAb-009 conjugates with 2.4, 3.5 and 5.5 CHX-A″ molecules were investigated. To investigate the effect of injected MORAb-009 dose on neutralizing the shed mesothelin in the circulation, biodistribution studies were performed after the intravenous co-injection of (111)In-labeled MORAb-009 (2.4 CHX-A″/MORAb-009) with three different doses: 0.2, 2 and 30 µg of MORAb-009. RESULTS: The tumor uptake in A431/K5 tumor was four times higher than that in A431 tumor, indicating that the tumor uptake in A431/K5 was mesothelin mediated. The conjugate with 5.5 CHX-A″ showed a lower isoelectric point (pI) and lower immunoreactivity (IR) than the 2.4 CHX-A″ conjugate. These differences were reflected in the biodistribution of the (111)In label. The (111)In-labeled MORAb-009 conjugated with 2.4 CHX-A″ produced higher tumor uptake and lower liver and spleen uptakes than the 5.5 CHX-A″ conjugate. The biodistribution studies also revealed that the tumor uptake was significantly affected by the injected MORAb-009 dose and tumor size. The 30-µg dose produced higher tumor uptake than the 0.2- and 2-µg doses, whereas the 30-µg dose produced lower liver and spleen uptakes than the 0.2-µg dose. CONCLUSION: This study demonstrates that the number of chelate conjugation and the injected dose are two important parameters to achieve high tumor and low non-target organ uptake of (111)In-labeled MORAb-009. This study also suggests that the injected dose of mAb could be individualized based on the tumor size or the blood level of shed antigen in a patient to achieve the ideal tumor-to-organ radioactivity ratios.

Importance of CYP2D3 in polymorphism of diazepam p-hydroxylation in rats.

Diazepam was metabolized to three primary metabolites, 3-hydroxy-diazepam, N-desmethyl-diazepam, and p-hydroxy-diazepam. Our previous studies reported metabolic position-specific inter- or intrastrain differences in diazepam metabolism among Sprague-Dawley, Brown Norway, Dark Agouti, and Wistar rats. Especially, there were marked ( approximately 300 fold) inter- or intrastrain differences in diazepam p-hydroxylation activity at low concentration of substrate. In this study, we investigated the enzyme that catalyzes diazepam p-hydroxylation. The activity toward diazepam p-hydroxylation was inhibited by anti-cytochrome P450 2D (CYP2D) antibody, suggesting that this activity was catalyzed by CYP2D isoforms. Comparing the expression levels of the CYP2D subfamily in liver microsomes from various strains of rats using anti-CYP2D2 antibody, we found that there was a band of protein that was consistent with the phenotype of diazepam p-hydroxylation. N-terminal amino acid sequences of the specific protein exactly corresponded to those of CYP2D3, indicating that CYP2D3 might be involved in diazepam p-hydroxylation. Moreover, using rat CYP2D isoforms expressed in yeast, we tested CYP2Ds to catalyze diazepam p-hydroxylation. CYP2D1 and CYP2D2 practically did not participate in diazepam metabolism. On the other hand, diazepam p-hydroxylation was catalyzed by CYP2D3. CYP2D4 had high activity toward diazepam N-desmethylation, but not p-hydroxylation. In conclusion, the polymorphic expression of CYP2D3 caused the inter- or intrastrain differences in diazepam p-hydroxylation among rat strains or individuals.

Strain differences in diazepam metabolism at its three metabolic sites in sprague-dawley, brown norway, dark agouti, and wistar strain rats.

Knowledge of strain differences in drug metabolism is important for the selection of animals for pharmacokinetic, pharmacodynamic, and toxicological studies. Hepatic microsomes from Sprague-Dawley (SD) and Brown Norway (BN) rats had 300-fold higher diazepam p-hydroxylation activity than Dark Agouti (DA) and Wistar (W) rats at a low diazepam concentration (3 microM). Kinetic studies indicated that diazepam p-hydroxylation in SD and BN rats proceeded with lower K(m) and higher V(max) values than it did in DA and W rats. However, the expression levels of cytochrome P450 CYP2D1, the reported enzyme for diazepam p-hydroxylation, did not cosegregate with the activity. These results suggest the presence of a new high-affinity diazepam p-hydroxylation enzyme other than CYP2D1 in SD and BN rats. DA rats showed 3- and 2-fold higher diazepam 3-hydroxylation and N-desmethylation activities, respectively, than the other rat strains. In agreement with this, DA rat liver microsomes had a higher expression of CYP3A2, which is responsible for diazepam 3-hydroxylation and partly responsible for N-desmethylation. Values of CL(int) (V(max)/K(m)) indicated that p-hydroxy-diazepam is the major metabolite in SD and BN rats, whereas 3-hydroxy-diazepam is the major metabolite in DA and W rats. The sum of the CL(int) in each strain was in the order of DA > SD = BN >> W. Strain differences in the pharmacodynamics of diazepam between SD and DA rats may be due to these differences in diazepam metabolism. We found that both the rate of elimination of diazepam and the major metabolic pathways in diazepam metabolism differed among the different rat strains due to polymorphic expression of the two enzymes involved in diazepam metabolism.

Short period exposure to di-(2-ethylhexyl) phthalate regulates testosterone metabolism in testis of prepubertal rats.

Exposure of pubertal rats to di-(2-ethylhexyl) phthalate (DEHP) for 14 days was reported to result in reduced testosterone (T) biosynthesis by altering androstenedione 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity. However, our study indicated that shorter period exposure of DEHP (100 or 1000 mg/kg for 5 days) to 4-week-old male rats did not affect the activity of 17beta-HSD, the rate-limiting enzyme of T biosynthesis in the testis. Testosterone 5alpha-reductase (T5alpha-R) activity in the testis was significantly enhanced, while aromatase mRNA was significantly reduced by increasing doses of DEHP. The expressions of cytochrome P450 (CYP) isoforms, CYP2C11 and CYP3A, in the testis increased along with their enzymatic activities, T16alpha- and T6beta-hydroxylation, respectively. Thus, the current study clearly indicates that the short period exposure to DEHP alters T metabolism through altering activities of T5alpha-R, aromatase and CYP2C11/3A2 in the testis of prepubertal rats, and that they are more sensitive marker enzymes to the DEHP exposure than those of biosynthetic enzymes of T from androstenedione.