Functional-structural decoupling in visual network is associated with cognitive decline in patients with type 2 diabetes mellitus: evidence from a multimodal MRI analysis.

Type 2 diabetes mellitus (T2DM) and cognitive dysfunction are highly prevalent disorders worldwide. Although visual network (VN) alteration and functional-structural coupling are potential warning factors for mild cognitive impairment (MCI) in T2DM patients, the relationship between the three in T2DM without MCI is unclear. Thirty T2DM patients without MCI and twenty-nine healthy controls (HC) were prospectively enrolled. Visual components (VC) were estimated by independent component analysis (ICA). Degree centrality (DC), amplitude of low frequency fluctuation (ALFF) and fractional anisotropy (FA) were established to reflect functional and structural characteristics in these VCs respectively. Functional-structural coupling coefficients were further evaluated using combined FA and DC or ALFF. Partial correlations were performed among neuroimaging indicators and neuropsychological scores and clinical variables. Three VCs were selected using group ICA. Deteriorated DC, ALFF and DC-FA coefficients in the VC1 were observed in the T2DM group compared with the HC group, while FA and ALFF-FA coefficients in these three VCs showed no significant differences. In the T2DM group, DC in the VC1 positively correlated with 2 dimensions in the California Verbal Learning Test, including Trial 4 and Total trial 1-5. The impaired DC-FA coefficients in the VC1 markedly affected the Total perseverative responses % of the Wisconsin Card Sorting Test. These findings indicate that DC and DC-FA coefficients in VN may be potential imaging biomarkers revealing early cognitive deficits in T2DM.

Commonalities and distinctions between the type 2 diabetes mellitus and Alzheimer's disease: a systematic review and multimodal neuroimaging meta-analysis.

Background: Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM) are aging related diseases with high incidence. Because of the correlation of incidence rate and some possible mechanisms of comorbidity, the two diseases have been studied in combination by many researchers, and even some scholars call AD type 3 diabetes. But the relationship between the two is still controversial. Methods: This study used seed-based d mapping software to conduct a meta-analysis of the whole brain resting state functional magnetic resonance imaging (rs-fMRI) study, exploring the differences in amplitude low-frequency fluctuation (ALFF) and cerebral blood flow (CBF) between patients (AD or T2DM) and healthy controls (HCs), and searching for neuroimaging evidence that can explain the relationship between the two diseases. Results: The final study included 22 datasets of ALFF and 22 datasets of CBF. The results of T2DM group showed that ALFF increased in both cerebellum and left inferior temporal gyrus regions, but decreased in left middle occipital gyrus, right inferior occipital gyrus, and left anterior central gyrus regions. In the T2DM group, CBF increased in the right supplementary motor area, while decreased in the middle occipital gyrus and inferior parietal gyrus. The results of the AD group showed that the ALFF increased in the right cerebellum, right hippocampus, and right striatum, while decreased in the precuneus gyrus and right superior temporal gyrus. In the AD group, CBF in the anterior precuneus gyrus and inferior parietal gyrus decreased. Multimodal analysis within a disease showed that ALFF and CBF both decreased in the occipital lobe of the T2DM group and in the precuneus and parietal lobe of the AD group. In addition, there was a common decrease of CBF in the right middle occipital gyrus in both groups. Conclusion: Based on neuroimaging evidence, we believe that T2DM and AD are two diseases with their respective characteristics of central nervous activity and cerebral perfusion. The changes in CBF between the two diseases partially overlap, which is consistent with their respective clinical characteristics and also indicates a close relationship between them. Systematic review registration: PROSPERO [CRD42022370014].

Abnormalities of cerebral blood flow and the regional brain function in Parkinson's disease: a systematic review and multimodal neuroimaging meta-analysis.

Background: Parkinson's disease (PD) is a neurodegenerative disease with high incidence rate. Resting state functional magnetic resonance imaging (rs-fMRI), as a widely used method for studying neurodegenerative diseases, has not yet been combined with two important indicators, amplitude low-frequency fluctuation (ALFF) and cerebral blood flow (CBF), for standardized analysis of PD. Methods: In this study, we used seed-based d-mapping and permutation of subject images (SDM-PSI) software to investigate the changes in ALFF and CBF of PD patients. After obtaining the regions of PD with changes in ALFF or CBF, we conducted a multimodal analysis to identify brain regions where ALFF and CBF changed together or could not synchronize. Results: The final study included 31 eligible trials with 37 data sets. The main analysis results showed that the ALFF of the left striatum and left anterior thalamic projection decreased in PD patients, while the CBF of the right superior frontal gyrus decreased. However, the results of multimodal analysis suggested that there were no statistically significant brain regions. In addition, the decrease of ALFF in the left striatum and the decrease of CBF in the right superior frontal gyrus was correlated with the decrease in clinical cognitive scores. Conclusion: PD patients had a series of spontaneous brain activity abnormalities, mainly involving brain regions related to the striatum-thalamic-cortex circuit, and related to the clinical manifestations of PD. Among them, the left striatum and right superior frontal gyrus are more closely related to cognition. Systematic review registration: https://www.crd.york.ac.uk/ PROSPERO (CRD42023390914).

A multi-channel collagen scaffold loaded with neural stem cells for the repair of spinal cord injury.

Collagen scaffolds possess a three-dimensional porous structure that provides sufficient space for cell growth and proliferation, the passage of nutrients and oxygen, and the discharge of metabolites. In this study, a porous collagen scaffold with axially-aligned luminal conduits was prepared. In vitro biocompatibility analysis of the collagen scaffold revealed that it enhances the activity of neural stem cells and promotes cell extension, without affecting cell differentiation. The collagen scaffold loaded with neural stem cells improved the hindlimb motor function in the rat model of T8 complete transection and promoted nerve regeneration. The collagen scaffold was completely degraded in vivo within 5 weeks of implantation, exhibiting good biodegradability. Rectal temperature, C-reactive protein expression and CD68 staining demonstrated that rats with spinal cord injury that underwent implantation of the collagen scaffold had no notable inflammatory reaction. These findings suggest that this novel collagen scaffold is a good carrier for neural stem cell transplantation, thereby enhancing spinal cord repair following injury. This study was approved by the Animal Ethics Committee of Nanjing Drum Tower Hospital (the Affiliated Hospital of Nanjing University Medical School), China (approval No. 2019AE02005) on June 15, 2019.

Decreased expression of TIPE2 in the eye under high-glucose conditions tested in vivo and in vitro.

AIMS: Inflammation is important in the development of angiogenesis diabetic retinopathy (DR). Anti-inflammation is promising strategy in early DR management. This study aimed to evaluate the level of tumour necrosis factor (TNF)-α-induced protein-8 like-2 (TIPE2), a formerly anti-inflammatory factor, under high-glucose conditions. METHODS: TIPE2 was detected in the ① retina from db/db and streptozotocin-induced diabetic mice; ② vitreous fluid of patients with proliferative diabetic retinopathy (PDR) and ③ mouse retinal microendothelial cells (RMEC) cultured in glucose of varying concentrations. In situ expression was evaluated by immunohistochemistry and immunofluorescence assay. The expression of protein was analysed by Western blot or ELISA and mRNA by qRT-PCR. RESULTS: TIPE2 was down-regulated in the retina of the mice with diabetes. TIPE2 was present in the cytoplasm of RMEC and down-regulated in high-glucose conditions in line with concentration and time. The expression of TIPE2 in the vitreous fluid of patients with PDR was significantly lower than that without diabetes. Silencing TIPE2 by an siRNA resulted in increased expression of vascular endothelial growth factor (a vital factor in the development of DR), TNF-α and IL-1ß. CONCLUSIONS: TIPE2 down-expressed and exerted anti-VEGF and anti-inflammatory function in the high-glucose environment. TIPE2 was verified to be involved in the process of DR and might be a potential regulator for DR development.

Melatonin Mitigates Oxazolone-Induced Colitis in Microbiota-Dependent Manner.

Levels of type 2 cytokines are elevated in the blood and intestinal tissues of ulcerative colitis (UC) patients in the active phase; this phenomenon indicates the participation of type 2 immune response in UC progression. The beneficial effects of melatonin in dextran sodium sulfate (DSS) and 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis models have been illustrated, but its role in the oxazolone (Oxa)-induced colitis model (driven by type 2 immune response) remains relatively unknown. We investigated the relationship between melatonin concentration and the severity of UC, revealing a significantly negative correlation. Subsequently, we investigated the effects of melatonin in Oxa-induced colitis mice and the potential underlying mechanisms. Administration of melatonin significantly counteracted body weight loss, colon shortening, and neutrophil infiltration in Oxa-induced colitis mice. Melatonin treatment mitigated Oxa-induced colitis by suppressing type 2 immune response. In addition, melatonin attenuated intestinal permeability by enhancing the expression of ZO-1 and occludin in colitis mice. Interestingly, the protective effect of melatonin was abolished when the mice were co-housed, indicating that the regulation of gut microbiota by melatonin was critical in alleviating Oxa-induced colitis. Subsequently, 16S rRNA sequencing was performed to explore the microbiota composition. Decreased richness and diversity of intestinal microbiota at the operational taxonomic unit (OTU) level resulted from melatonin treatment. Melatonin also elevated the abundance of Bifidobacterium, a well-known probiotic, and reduced proportions of several harmful bacterial genera, such as Desulfovibrio, Peptococcaceae, and Lachnospiraceae. Fecal microbiota transplantation (FMT) was used to explore the role of microbiota in the function of melatonin in Oxa-induced colitis. Microbiota transplantation from melatonin-treated mice alleviated Oxa-induced colitis, suggesting that the microbiome participates in the relief of Oxa-induced colitis by melatonin. Our findings demonstrate that melatonin ameliorates Oxa-induced colitis in a microbiota-dependent manner, suggesting the therapeutic potential of melatonin in treating type 2 immunity-associated UC.

Mobile Health to Maintain Continuity of Patient-Centered Care for Chronic Kidney Disease: Content Analysis of Apps.

BACKGROUND: Chronic kidney disease (CKD) is a global health problem with a high economic burden, which is particularly prevalent in Taiwan. Mobile health apps have been widely used to maintain continuity of patient care for various chronic diseases. To slow the progression of CKD, continuity of care is vital for patients' self-management and cooperation with health care professionals. However, the literature provides a limited understanding of the use of mobile health apps to maintain continuity of patient-centered care for CKD. OBJECTIVE: This study identified apps related to the continuity of patient-centered care for CKD on the App Store, Google Play, and 360 Mobile Assistant, and explored the information and frequency of changes in these apps available to the public on different platforms. App functionalities, like patient self-management and patient management support for health care professionals, were also examined. METHODS: We used the CKD-related keywords "kidney," "renal," "nephro," "chronic kidney disease," "CKD," and "kidney disease" in traditional Chinese, simplified Chinese, and English to search 3 app platforms: App Store, Google Play, and 360 Mobile Assistant. A total of 2 reviewers reached consensus on coding guidelines and coded the contents and functionalities of the apps through content analysis. After coding, Microsoft Office Excel 2016 was used to calculate Cohen kappa coefficients and analyze the contents and functionalities of the apps. RESULTS: A total of 177 apps related to patient-centered care for CKD in any language were included. On the basis of their functionality and content, 67 apps were recommended for patients. Among them, the most common functionalities were CKD information and CKD self-management (38/67, 57%), e-consultation (17/67, 25%), CKD nutrition education (16/67, 24%), and estimated glomerular filtration rate (eGFR) calculators (13/67, 19%). In addition, 67 apps were recommended for health care professionals. The most common functionalities of these apps were comprehensive clinical calculators (including eGFR; 30/67; 45%), CKD medical professional information (16/67, 24%), stand-alone eGFR calculators (14/67, 21%), and CKD clinical decision support (14/67, 21%). A total of 43 apps with single- or multiple-indicator calculators were found to be suitable for health care professionals and patients. The aspects of patient care apps intended to support self-management of CKD patients were encouraging patients to actively participate in health care (92/110, 83.6%), recognizing and effectively responding to symptoms (56/110, 50.9%), and disease-specific knowledge (53/110, 48.2%). Only 13 apps contained consulting management functions, patient management functions or teleconsultation functions designed to support health care professionals in CKD patient management. CONCLUSIONS: This study revealed that the continuity of patient-centered care for CKD provided by mobile health apps is inadequate for both CKD self-management by patients and patient care support for health care professionals. More comprehensive solutions are required to enhance the continuity of patient-centered care for CKD.

A Content Analysis of Mobile Apps for Chronic Kidney Disease Patient Care: Searching in English and Chinese.

Chronic kidney disease (CKD) is a global public health issue. Mobile technology is pervasive and widely used in chronic disease care. More and more, CKD mobile applications (apps) can be found on popular mobile application platforms, especially in Chinese. We aim to explore current mobile apps for CKD patient care (searching in English and Chinese) through content analysis to identify the app functions that health professionals can use in CKD patient care and to help CKD patients with self-management.

Anti-inflammatory TIPE2 inhibits angiogenic VEGF in retinal pigment epithelium.

Choroidal neovascularization (CNV) is a pathological feature which commonly occurs in ocular diseases. This condition is characterised by vasculogenesis and angiogenesis underlying the neuroretina, with retinal pigment epithelium (RPE) and choroid as main targets. Inflammation and immunity are crucial in the early development of CNV. Tumour necrosis factor (TNF) α-induced protein-8 like-2 (TIPE2 or TNFAIP8L2), a recently identified gene, is a negative regulator of innate and adaptive immunity which participates in inflammatory homeostasis. We determined the expression of TIPE2 in normal and inflamed RPE cells, and evaluated the relationship of TIPE2 with factors associated with inflammation and angiogenesis. TIPE2 is present in both the cytoplasm and nucleus of human RPE cells and is down-regulated in the inflammatory state with decreased cell viability. Knock-down of TIPE2 by a specific short interfering RNA increases the expression levels of TNF-α, IL-1ß and angiogenic vascular endothelial growth factor (VEGF), particularly under the stimulation of lipopolysaccharide. In consideration of the vital role of VEGF in the final stage of neovascularization, the anti-inflammatory TIPE2 is also anti-angiogenic and may participate in CNV formation.

Production of shikimic acid from Escherichia coli through chemically inducible chromosomal evolution and cofactor metabolic engineering.

BACKGROUND: Shikimic acid (SA) produced from the seeds of Chinese star anise (Illicium verum) is a key intermediate for the synthesis of neuraminidase inhibitors such as oseltamivir (Tamiflu®), an anti-influenza drug. However, plants cannot deliver a stable supply of SA. To avoid the resulting shortages and price fluctuations, a stable source of affordable SA is required. Although recent achievements in metabolic engineering of Escherichia coli strains have significantly increased SA productivity, commonly-used plasmid-based expression systems are prone to genetic instability and require constant selective pressure to ensure plasmid maintenance. Cofactors also play an important role in the biosynthesis of different fermentation products. In this study, we first constructed an E. coli SA production strain that carries no plasmid or antibiotic marker. We then investigated the effect of endogenous NADPH availability on SA production. RESULTS: The pps and csrB genes were first overexpressed by replacing their native promoter and integrating an additional copy of the genes in a double gene knockout (aroK and aroL) of E. coli. The aroG(fbr), aroB, aroE and tktA gene cluster was integrated into the above E. coli chromosome by direct transformation. The gene copy number was then evolved to the desired value by triclosan induction. The resulting strain, E. coli SA110, produced 8.9-fold more SA than did the parental strain E. coli (ΔaroKΔaroL). Following qRT-PCR analysis, another copy of the tktA gene under the control of the 5P(tac) promoter was inserted into the chromosome of E. coli SA110 to obtain the more productive strain E. coli SA110. Next, the NADPH availability was increased by overexpressing the pntAB or nadK genes, which further enhanced SA production. The final strain, E. coli SA116, produced 3.12 g/L of SA with a yield on glucose substrate of 0.33 mol/mol. CONCLUSION: An SA-producing E. coli strain that carries neither a plasmid nor an antibiotic marker was constructed by triclosan-induced chromosomal evolution. We present the first demonstration that increasing NADPH availability by overexpressing the pntAB or nadK genes significantly enhances SA production.

Chromosomal evolution of Escherichia coli for the efficient production of lycopene.

BACKGROUND: Plasmid-based overexpression of genes has been the principal strategy for metabolic engineering. However, for biotechnological applications, plasmid-based expression systems are not suitable because of genetic instability, and the requirement for constant selective pressure to ensure plasmid maintenance. RESULTS: To overcome these drawbacks, we constructed an Escherichia coli lycopene production strain that does not carry a plasmid or an antibiotic marker. This was achieved using triclosan-induced chromosomal evolution, a high gene copy expression system. The engineered strain demonstrated high genetic stability in the absence of the selective agent during fermentation. The replacement of native appY promoter with a T5 promoter, and the deletion of the iclR gene in E. coli CBW 12241 further improved lycopene production. The resulting strain, E. coli CBW 12241(ΔiclR, PT5-appY), produced lycopene at 33.43 mg per gram of dry cell weight. CONCLUSIONS: A lycopene hyper-producer E. coli strain that does not carry a plasmid or antibiotic marker was constructed using triclosan-induced chromosomal evolution. The methods detailed in this study can be used to engineer E. coli to produce other metabolites.

[Effects of P 311 on the migration of epidermal stem cells in mice with superficial partial-thickness burn and injured cell model in vitro].

OBJECTIVE: To study effects of P311 on the migration of epidermal stem cells (ESCs) in mice with superficial partial-thickness burn and injured cell model in vitro and to explore the mechanism. METHODS: (1) Eighteen male C(57) BL/6 mice were used. Fifteen of them were inflicted with superficial partial-thickness burn on the back. In three injured mice wound tissue and skin of wound edge were obtained at post burn hour (PBH) 6, 12, 24, 48, 72 respectively. The rest three mice were used as normal control, and samples were harvested with the same method as above. The expressions of P311 in harvested samples were assessed with biotin-streptavidin-peroxidase (SP) staining. (2) Six newly born C(57) BL/6 mice were intraperitoneally injected with 50 µg/g BrdU (two times a day) for three days for ESCs-labelling. Seven weeks later, the mice were inflicted with superficial partial-thickness burn on the back. Serial slices of burn wound tissue were prepared at PBH 72 and immunohistochemically stained with SP for observation of the co-localization of BrdU-positive ESCs and P311-positive cells. (3) The empty vector pAdEasy-enhanced green fluorescence protein (EGFP) and the adenovirus P311-expressing vector named pAdEasy-EGFP-P311 were constructed and packed. Human ESCs were isolated by the method of rapid adhesion to collagen IV. After being divided into P311 high-expressing group (n = 3) and EGFP control group (n = 3), the ESCs in two groups were respectively infected by pAdEasy-EGFP-P311 and pAdEasy-EGFP. Scratching assay was performed on ESCs in both groups after they were treated by mitomycin C for 2 hours. The remaining area within the fixed range was measured at post scratching hour (PSH) 0, 24, 48, and 72, and the wound-area healing rate was calculated. Data were processed with independent samples t test. RESULTS: (1) Expression amount of P311 was different in different parts of wound at different time points after burn. Expression amount of P311 in the newly formed epidermis and hair follicle of wound increased along with prolongation of time. Expression amount of P311 in the epidermis and hair follicle of wound edge peaked at PBH 12 and then decreased to normal levels at PBH 72. (2) Co-localization of BrdU-positive ESCs and P311-positive cells was observed in the new epidermal layer of wound tissue of mice, where ESCs were labeled by BrdU. (3) At PSH 48 and 72, wound-area healing rate was obviously higher in P311 high-expressing group [(69 ± 31)%, (89 ± 26)%] than in EGFP control group [(35 ± 12)%, (46 ± 31)%, with t values respectively -2.336, -2.611, P values all below 0.05]. CONCLUSIONS: P311 may promote the migration of ESCs both in rats with superficial partial-thickness burns and in injured cell model in vitro, and it may play an important role in wound healing.

[Biologic effect of nitric oxide on human epidermal stem cells in vitro].

OBJECTIVE: To observe the effect of nitric oxide (NO) on adhesion, proliferation, and migration of human epidermal stem cells (ESC) in vitro. METHODS: ESC were isolated and cultured by the modified method of rapid attachment to type IV collagen. (1) Morphology of cells was observed under inverted phase-contrast microscope. Expression levels of integrin ß(1) and cytokeratin 19 (CK19) of cells were determined by Western blotting and immunofluorescence staining. (2) After being treated with scratching, ESC adhered to the wall was respectively treated with nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP) in the concentration of 1, 10, 100, 500 µmol/L. ESC without treatment of SNAP was used as control. The migration rate of ESC was detected at post scratching hour (PSH) 12 and 24. The chemotaxis of ESC (treated with SNAP in above-mentioned concentration) was tested by Transwell assay, and the transferred cell number was counted. (3) ESC was respectively treated with SNAP in the concentration of 10, 100, 500 µmol/L for 1 h. ESC without treatment of SNAP was used as control. The adhesion of ESC was detected with adhesion test, and the inhibition rate of adhesion was calculated. The proliferation of ESC (denoted as absorbance value) was determined by microplate reader at post-treatment hour (PTH) 0, 12, 24, 48. Data were processed with one-way analysis of variance and Dunnett t test. RESULTS: (1) Small clone formed on post culture days (PCD) 5 to 9. On PCD 10 to 14, cell proliferation sped up. CK19 and integrin ß(1) were detected to be expressed in the isolated cells. The cells were identified as ESC. (2) Compared with that of ESC without treatment of SNAP [(35.7 ± 0.3)%, (45.7 ± 5.0)%], migration of ESC treated with SNAP in the concentration from 1 to 100 µmol/L was promoted at PSH 12 and 24. Migration rates of ESC treated with 100 µmol/L SNAP were the highest [respectively (48.8 ± 2.7)%, (82.1 ± 15.8)%, with t value respectively 8.34, 5.10, P values both below 0.01]. The number of ESC transferred to membrane after being treated with 100 µmol/L SNAP was significantly larger than that of ESC without treatment of SNAP (t = 9.24, P = 0.00). (3) Absorbance values of ESC treated with 100, 500 µmol/L SNAP were obviously higher than that of ESC without treatment of SNAP (with t value respectively 4.30, 4.67, P values both equal to 0.00). Proliferation of ESC treated with 100, 500 µmol/L SNAP was obviously stronger than that of cells without treatment of SNAP at PTH 24, 48 (with t values from 2.84 to 8.17, P values all below 0.05). CONCLUSIONS: Exogenous NO in suitable concentration can promote the migration of human ESC. Exogenous NO can inhibit the adhesion and promote the proliferation of human ESC in vitro.

Induction and inhibition of cytochromes P450 by the St. John's wort constituent hyperforin in human hepatocyte cultures.

St. John's wort extract (SJW) (Hypericum perforatum L.) is among the most commonly used herbal medications in the United States. The predominance of clinical reports indicates that SJW increases the activity of cytochrome P450 3A4 (CYP3A4) enzyme and reduces plasma concentrations of certain drugs. Although the inductive effect of SJW on CYP3A4 is clear, other reports indicate that SJW constituents may have, to a small degree, some enzyme inhibitory effects. Therefore, we sought to study the induction and inhibition effects of the constituents of SJW on CYP3A4 in the human hepatocyte model. Moreover, most research has focused on the induction of CYP3A4 by SJW with little attention paid to other prominent drug-metabolizing enzymes such as CYP1A2, CYP2C9, and CYP2D6. To examine the effects of SJW on CYP1A2, CYP2C9, CYP2D6, as well as CYP3A4, hepatocytes were exposed to hyperforin and hypericin, the primary constituents of SJW extract. Hepatocytes treated with hypericin or hyperforin were exposed to probe substrates to determine enzyme activity and protein and RNA harvested. Hyperforin treatment resulted in significant increases in mRNA, protein, and activity of CYP3A4 and CYP2C9, but had no effect on CYP1A2 or CYP2D6. Acute administration of hyperforin at 5 and 10 microM 1 h before and along with probe substrate inhibited CYP3A4 activity. Hypericin had no effect on any of the enzymes tested. These results demonstrate that with chronic exposure, the inductive effect of SJW on drug-metabolizing enzymes predominates, and human hepatocyte cultures are a versatile in vitro tool for screening the effect of herbal products on cytochrome P450 enzymes.

Gender differences in microsomal metabolic activation of hepatotoxic clivorine in rat.

The gender differences in the in vitro microsomal metabolic activation of hepatotoxic clivorine, a representative naturally occurring hepatotoxic otonecine type pyrrolizidine alkaloid, in Sprague-Dawley rats and their relation to the gender differences in susceptibility to clivorine intoxication were reported in the present study. Clivorine-induced liver damage in the male rat via metabolic activation to form the reactive pyrrolic ester followed by covalent binding to liver tissue constituents has been reported previously by our research group. The present study demonstrated, for the first time, that cytochromes p450 3A1 and 3A2, which are constitutively expressed in the male rat, might play a significant role in the metabolic activation of clivorine in the rat. Thus, in the male rat, the metabolic activation by liver microsomes to form the reactive pyrrolic ester was found as the only direct metabolic pathway of clivorine followed by subsequent formation of the toxic tissue-bound pyrroles leading to hepatotoxicity. In the case of the female rat, a less significant metabolic activation was observed, whereas the formations of two novel nonpyrrolic metabolites were determined as the predominant biotransformations. None of the four cDNA-expressed rat enzymes (cytochrome p450 2C12, 2E1, 3A1, 3A2) tested could catalyze the formation of these two new metabolites. Furthermore, the female rat (LD(50) = 114 +/- 9 mg/kg, i.p.) was found to be significantly less susceptible to clivorine intoxication than the male rat (LD(50) = 91 +/- 3 mg/kg, i.p.). Therefore, the results suggested that a significantly lower metabolic activation due to the lack of cytochrome p450 3A1 and p450 3A2 activities mainly accounted for the smaller susceptibility of the female rat to clivorine intoxication.

Species differences in the in vitro metabolic activation of the hepatotoxic pyrrolizidine alkaloid clivorine.

Clivorine is a representative naturally occurring hepatotoxic otonecine-type pyrrolizidine alkaloid. Our previous study has demonstrated that clivorine induces liver damage via metabolic activation to form the reactive pyrrolic ester followed by covalent binding to liver tissue constituents. The present study investigated species differences in the in vitro metabolic activation of clivorine in the male rat and guinea pig of both sexes. In the male rat, the activation of clivorine to form the reactive pyrrolic ester was found as the only metabolic pathway. Moreover, the toxic tissue-bound pyrroles and four isolatable metabolites identified, namely DHR, 7-GSH-DHR, 7,9-diGSH-DHR, and clivoric acid, were all generated from further metabolism of this reactive intermediate. In the case of both sexes of guinea pig, the same activation was observed as the minor biotransformation, while an additional metabolic pathway, a direct hydrolysis of clivorine to form novel clivopic acid was identified as the predominant detoxification pathway. Furthermore, the formation rates for the toxic tissue-bound pyrroles and less toxic DHR were significantly slower and higher, respectively, compared with those in the male rat. In addition, the formation of the reactive pyrrolic ester was mediated by the CYP3A subfamily in both animals, while carboxylesterases might be responsible for the detoxification hydrolysis in guinea pig. The results suggest that the higher metabolic rates for detoxification hydrolyses and the lower formation rate for the toxic tissue-bound pyrroles play the key roles in guinea pig resistance to clivorine intoxication. Therefore, the male rat and guinea pig should be the suitable animal models for further studies of bioactivation and deactivation of otonecine-type PA, respectively.