Control Rat Serum Research Articles

Osteoarthritis rat serum-derived extracellular vesicles aggravate osteoarthritis development by inducing NLRP3-mediated pyroptotic cell death and cellular inflammation.

Osteoarthritis (OA), degenerative joint disease, is the most prevalent form of arthritis worldwide. Besides its substantial burden on society, the high OA morbidity greatly diminishes patients' quality of life. According to recent research, patients-derived serum extracellular vesicles (EVs) are critically involved in sustaining the corresponding disease progression. However, limited research has fully explored the specific functions and molecular mechanisms of OA serum-derived EVs in disease progression. Consequently, we aimed to investigate the underlying mechanism of OA rats-derived serum EVs in regulating OA progression. Before constructing the exosome-cell co-culture system, EVs were extracted from OA and control rat serum and co-cultured with bone marrow mesenchymal stem cells (BM-MSCs). Western blotting (WB), RT-qPCR, and enzyme-linked immunosorbent assay (ELISA) results revealed that OA rat serum-derived EVs upregulated cell pyroptosis-related markers, including nod-Like receptor protein-3 (NLRP3), apoptosis-associated speck-like protein (ASC), gasdermin D (GSDMD), and cleaved caspase-1. The OA rat-EVs also induced the release of LDH and inflammatory cytokines, including interleukin (IL)-1β, IL-18, IL-6, and TNF-α. Additional experiments revealed that OA rat-EVs delivered miR-133a-3p to BM-MSCs and upregulated miR-133a-3p to degrade sirtuin 1 (SIRT1), and activating the downstream NF-κB signaling pathway. Furthermore, the rescuing experiments confirmed that silencing SIRT1 abrogated the miR-133a-3p-induced protective effects in OA-EVs-treated BM-MSCs. In conclusion, OA rats-derived miR-133a-3p-containing EVs modulated the downstream SIRT1/NF-κB pathway-mediated pyroptotic cell death and inflammation in OA. In other words, this study confirmed the role and underlying mechanisms by which OA-associated serum EVs regulate pyroptosis and inflammation response in OA development.

Analysis of phytochemical constituents of zuogui wan in rat serum and its effects on early embryonic development of mice

Objective: Zuogui Wan (ZGW) has been used as a typical prescription for tonifying kidney essence in traditional Chinese medicine. The objective of this study is to elucidate the phytochemical constituents of ZGW-treated rat serum (ZGWRS) using ultra-performance liquid chromatography-electrospray ionization/quadrupole-time-of-flight high-definition mass spectrometry (UPLC-ESI-Q-TOF-MS). Methods: ZGW was administered to rats, and the phytochemical constituents in rat serum were determined using UPLC-ESI-Q-TOF-MS. MetaboLynx analysis in negative ion mode was adopted to characterize the chemical constituents of ZGWRS. Orthogonal partial least squares discriminant analysis was applied for the discovery of constituents of ZGW that entered the serum of rats. The fertilized eggs collected from the same experiment were randomly divided into four groups, including the normal, 40 mmol/L glucose, 40 mmol/L glucose 5% control rat serum, and 40 mmol/L glucose 5% ZGWRS groups. They were cultivated at 37°C and 5% CO2in a saturated humidity CO2incubator. The blastocyst rate and two-cell rate were used to evaluate the effects of ZGWRS on embryonic development. Results: Thirteen constituents were identified in the ZGWRS, among which sweroside, loganin, morroniside, loganic acid, and 8-epiloganic acid were from Fructus Corni (Shan Zhu Yu). 5-Hydroxymethyl-2-furfural-glucuronide, 2-H-5-hydroxymethyl-2-furfural-glucuronide, 3-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxylic acid, and β-D-ribofuranuronic acid methyl ester triacetate were from Radix Rehmanniae Preparata (Shu Di Huang). Coumaric acid was from Fructus Lycii (Gou Qi Zi). Kaempferol-3-beta-O-glucuronide and cuscutamine were from Semen Cuscutae (Tu Si Zi). The embryonic development was significantly inhibited using 40 mmol/L glucose. Compared with the normal group, the blastocyst rate of the glucose group was decreased. The blastocyst rate of the 40 mmol/L glucose 5% ZGWRS group was significantly higher than that of the glucose group, indicating that ZGWRS negates the effect of glucose on mouse embryonic development. Conclusion: The results verified that a rapid and robust UPLC-ESI-Q-TOF-MS-based platform had been successful for identifying multiple constituents of ZGW. ZGWRS is rich in active constituents of iridoid glycosides. The results of this study also showed that ZGWRS could negate the effect of glucose on mouse embryonic development.

Using the human CYP26A1 gene promoter as a suitable tool for the determination of RAR-mediated retinoid activity.

We have used a shortened construct form of the CYP26A1 gene promoter, in a promoter-less vector with either luciferase (known as E4) or a red fluorescent protein, RFP (known as E4.2) as the reporter gene and examined their responses to retinoids in transfected HepG2 and HEK293T cells. The promoter responded linearly to a wide concentration range of at-RA in cells cotransfected with retinoic acid receptors (RAR). The promoter also responded quantitatively to retinol and various other retinoids. An isolated clonal line of HEK293T cells that was permanently transfected with the promoter driving the expression of RFP responded to both at-RA and retinol, and the responses could be measured by fluorescence microscopy and flow cytometry. The promoter was also used to assess the retinoid activity of 3 novel synthetic retinoid analogues. Among them, EC23 was shown to be more potent than at-RA at lower concentrations and also more stable than at-RA. The promoter was also used to estimate the retinoid activities of intact rat serum samples as well as extracts of rat liver and lung, using retinol and at-RA as the reference standards. The retinoid activities could be measured in control rat serum samples and were increased in the serum of at-RA-treated rats. The total retinol and at-RA levels in the rat liver and lung samples determined by this promoter-based assay were compared with total retinol levels determined by the UPLC as the conventional methods. This system should offer a biologically-based alternative to mass-based retinoid analysis.

CYP26A1 gene promoter is a useful tool for reporting RAR-mediated retinoid activity

Of numerous genes regulated by retinoic acid (RA), CYP26A1 is the most inducible gene by RA. In this study, we have used a shortened construct form, E4, of the CYP26A1 gene promoter, in a promoter-less vector with either luciferase or red fluorescent protein (RFP) as the reporter gene and have tested its responses to retinoids in transfected HepG2 and HEK293T cells. The promoter responded linearly to a wide concentration range of RA in cells cotransfected with retinoic acid receptors. It also responded quantitatively to retinol and other retinoids. An isolated clonal line of HEK293T cells permanently transfected with the promoter driving the expression of RFP responded to both RA and retinol, and the responses could be measured by fluorescence microscopy and flow cytometry. The promoter was used to assess the retinoid activity of 3 novel synthetic retinoid analogues, as well as of the intact serum samples of rats. Among the synthetic retinoid analogues tested, EC23 is more potent than RA at lower concentrations and was more stable than RA. The retinoid activities could be measured in control rat serum samples and were increased in the serum of RA-treated rats. This system offers a biologically-based alternative to mass-based retinoid analysis.

Effect of Comus officinalis glucosides on serum levels of interleukin-18, transforming growth factor-β1 and pathological changes in kidney tissues in IgA nephropathy rat models

Objective To study the mechanism of Comus officinalis glucosides treatment of IgA nephropathy. Methods Fifty SD rats were randomly divided into the control group, the IgAN model group, the Dexamethasone group, the Comus officinalis glucosides in the high-dose group and in the low-dose group.The IgA nephropathy rat models were established by feeding bovine serum albumin (BSA), subcutaneous injection of carbon tetrachloride (CCl4), castor oil and injecting tail vein with lipopolysaccharide (LPS). After 10 weeks of administration, the urine red blood cell count, UTP in each phase were observed after the rat models were successfully established.Then Dexamethasone group and Cornus officinalis total glucoside group were given medicine for 8 weeks, and urine or serum content of interleukin-18 (IL-18), transforming growth factor-β1 (TGF-β1) in different groups of rats and pathological changes of kidneys were detected. Results Compared with the IgAN model group, 24 h-UTP of each group decreased and Comus officinalis glucosides in high-dose group decreased extraordinary[(8.8±3.2) mg/24 h vs.(35.2±5.6) mg/24 h, t=10.511, P=0.000]. Compared with the IgAN model group, urine red blood cell count decreased significantly in the high-dose group of Comus officinalis glucosides[(148.2±41.8)个/μL vs.(683.8±24.6)个/μL, t=10.909, P=0.000]; but urine red blood cell count in the Dexamethasone group declined slightly, but there was no statistically significant difference(t=3.014, P=0.061). Compared with the control group, IL-18, TGF-β1, and rat urine or serum of IgAN model group was significantly elevated (all P<0.05). Compared with IgAN model group, IL-18, TGF-β1 of the high-dose group of Comus officinalis glucosides in rats urine or serum decreased significantly(all P<0.05). The effect of high-dose group of Cornus officinalis glucosides on the pathological changes in kidney tissues was the most significant. Conclusions The mechanism of Comus officinalis glucosides may postpone renal lesion that can be associated with the down-regulation of the expression of IL-18, TGF-β1 and the inhibition of the deposition of IgA in the renal tissue.Comus officinalis glucosides can inhibit the proliferation of mesangial cells, tubular atrophy and interstitial fibrosis in IgAN rats, which has a certain curative effect on IgAN rats. Key words: Comus officinalis glucosides; IgA nephropathy rats; Interleukin-18; Transforming growth factor-β1; Renal tissue

Simulating vasogenic brain edema using chronic VEGF infusion.

OBJECTIVE To study peritumoral brain edema (PTBE), it is necessary to create a model that accurately simulates vasogenic brain edema (VBE) without introducing a complicated tumor environment. PTBE associated with brain tumors is predominantly a result of vascular endothelial growth factor (VEGF) secreted by brain tumors, and VEGF infusion alone can lead to histological blood-brain barrier (BBB) breakdown in the absence of tumor. VBE is intimately linked to BBB breakdown. The authors sought to establish a model for VBE with chronic infusion of VEGF that can be validated by serial in-vivo MRI and histological findings. METHODS Male Fischer rats (n = 182) underwent stereotactic striatal implantation of MRI-safe brain cannulas for chronic infusion of VEGF (2-20 µg/ml). Following a preinfusion phase (4-6 days), the rats were exposed to VEGF or control rat serum albumin (1.5 µl/hr) for as long as 144 hours. Serial MRI was performed during infusion on a high-field (9.4-T) machine at 12-24, 24-36, 48-72, and 120-144 hours. Rat brains were then collected and histological analysis was performed. RESULTS Control animals and animals infused with 2 µg/ml of VEGF experienced no neurological deficits, seizure activity, or abnormal behavior. Animals treated with VEGF demonstrated a significantly larger volume (42.90 ± 3.842 mm3) of T2 hyper-attenuation at 144 hours when compared with the volume (8.585 ± 1.664 mm3) in control animals (mean difference 34.31 ± 4.187 mm3, p < 0.0001, 95% CI 25.74-42.89 mm3). Postcontrast T1 enhancement in the juxtacanalicular region indicating BBB breakdown was observed in rats undergoing infusion with VEGF. At the later time periods (120-144 hrs) the volume of T1 enhancement (34.97 ± 8.99 mm3) was significantly less compared with the region of edema (p < 0.0001). Histologically, no evidence of necrosis or inflammation was observed with VEGF or control infusion. Immunohistochemical analysis demonstrated astrocyte activation, vascular remodeling, and increased claudin-5 expression in juxtacanalicular regions. Aquaporin-4 expression was increased in both control and VEGF animals in the juxtacanalicular regions. CONCLUSIONS The results of this study show that chronic brain infusion of VEGF creates a reliable model of VBE. This model lacks necrosis and inflammation that are characteristic of previous models of VBE. The model allows for a precise investigation into the mechanism of VBE formation. The authors also anticipate that this model will allow for investigation into the mechanism of glucocorticoid action in abrogating VBE, and to test novel therapeutic strategies targeting PTBE.

Metal Ion Imbalance-Related Oxidative Stress Is Involved in the Mechanisms of Liver Injury in a Rat Model of Chronic Aluminum Exposure.

The objective of the study is to investigate the effects of chronic aluminum overload on rat liver function and its induction of pathological changes in metal ion levels and oxidative stress in hepatic tissues. Wistar rats were intragastrically administered aluminum gluconate (200mg Al(3+)/Kg) once a day, 5days a week, for 20weeks. HE staining was used to visualize pathological changes in rat liver tissue. A biochemical method was adopted to detect ALT, AST, ALP, and GGT levels, as well as liver SOD activity and blood plasma MDA content. A plasma atomic emission spectrophotometer was used to detect Al, Mn, Fe, Zn, and Cu ion contents in liver tissue. Our results showed obvious vacuolar degeneration, granular degeneration, and spotty necrosis in chronic Al-overload rat hepatocytes. The levels of ALT, AST, ALP, and GGT were significantly increased. Liver SOD activity was significantly decreased, and MDA content was significantly increased. In Al-overload rat liver, Al, Mn, Fe, and Cu contents were significantly increased, and in Al-overload rat serum, Mn, Fe, Zn, and Cu contents were significantly decreased. However, the Al level in Al-overload rat serum was not significantly different from that in control rat serum. These results suggest that chronic aluminum overload causes obvious damage to rat liver and causes imbalances in Al, Mn, Fe, Zn, and Cu levels in rat liver and serum. Metal ion imbalance-related oxidative stress may be involved in the mechanism of chronic liver injury caused by aluminum overload.

Comparison of Life-Stage-Dependent Internal Dosimetry for Bisphenol A, Ethinyl Estradiol, a Reference Estrogen, and Endogenous Estradiol to Test an Estrogenic Mode of Action in Sprague Dawley Rats

Bisphenol A (BPA) was administered by gavage (2.5-300,000 μg/kg body weight (bw)/day) to pregnant Sprague Dawley dams, newborn pups, and continuing into adulthood. Aglycone (i.e., unconjugated and active) and conjugated (i.e., inactive) BPA were evaluated by liquid chromatography electrospray tandem mass spectrometry (LC-ES/MS/MS) in serum to better interpret toxicological endpoints measured in the study. Ethinyl estradiol (EE2, 0.5 and 5 μg/kg bw/day) and the endogenous hormones, 17β-estradiol (E2) and testosterone, were similarly evaluated. Mean BPA aglycone levels in vehicle and naïve control rat serum (0.02-0.5 ng/ml) indicated sample processing artifact, consistent with literature reports of a propensity for postexposure blood contamination by BPA. Direct measurements of BPA-glucuronide in vehicle and naïve control serum (2-10nM) indicated unintentional exposure and metabolism at levels similar to those produced by 2.5 μg/kg bw/day BPA (7-10nM), despite careful attention to potential BPA inputs (diet, drinking water, vehicle, cages, bedding, and dust) and rigorous dosing solution certification and delivery. The source of this exposure could not be identified, but interpretation of the toxicological effects, observed only at the highest BPA doses, was not compromised. Internal exposures to BPA and EE2 aglycones were highest in young rats. When maximal serum concentrations from the two highest BPA doses and both EE2 doses were compared with concurrent levels of endogenous E2, the ERα binding equivalents were similar to or above those of endogenous E2 in male and female rats of all ages tested. Such evaluations of estrogenic internal dosimetry and comprehensive evaluation of contamination impact should aid in extrapolating risks from human BPA exposures.

Effects of Chronic Renal Failure on Brain Drug Transporters in Rats

Studies demonstrated that chronic renal failure (CRF) affects the expression and activity of intestinal, hepatic, and renal drug transporters. Such drug transporters are expressed in brain cells and at the blood-brain barrier (BBB), where they limit the entry and distribution of drugs in the brain. Perturbations in brain drug transporter equilibrium by CRF could lead to central drug toxicity. This study evaluates how CRF affects BBB drug transporters using a 5/6 nephrectomized rat model. Protein and mRNA expression of influx transporters [organic anion-transporting polypeptide (Oatp), organic anion transporter (Oat)] and efflux transporters [P-glycoprotein (P-gp), multidrug resistance-related protein (Mrp), breast cancer resistance protein (Bcrp)] were measured in CRF and control rat brain. Intracerebral accumulation of radiolabeled benzylpenicillin, digoxin, doxorubicin, and verapamil was used to evaluate BBB drug permeability. Protein expression of the transporters was evaluated in rat brain endothelial cells (RBECs) and astrocytes incubated with control and CRF rat serum. We demonstrated significant decreases (30-50%) in protein and mRNA levels of Bcrp, Mrp2 to -4, Oat3, Oatp2 and -3, and P-gp in CRF rat brain biopsies, as well as in astrocytes and RBECs incubated with CRF serum. These decreases did not correlate with in vivo changes because BBB permeability of benzylpenicillin was decreased by 30% in CRF rats, whereas digoxin, doxorubicin, and verapamil permeabilities were unchanged. It thus seems that even with decreased drug transporters, BBB integrity and function is conserved in CRF.

Simultaneous determination of serum lathosterol and cholesterol by semi-micro high-performance liquid chromatography with electrochemical detection

A simple method has been developed for the simultaneous determination of lathosterol and cholesterol by high-performance liquid chromatography with electrochemical detection (HPLC–ECD). Lathosterol was found to be electrochemically oxidized and its current peak height was linearly related to the amount of lathosterol injected, ranging from 0.15 μmol/L to 300 μmol/L ( r = 0.995). Similar results were obtained with cholesterol from 15 μmol/L to 600 μmol/L ( r = 0.995). The separation was carried out with an ODS column, acetonitrile containing 30 mmol/L lithium perchlorate as a mobile phase, and an applied potential at +2.8 V vs. Ag/AgCl. The detection limit ( S/ N = 3) of lathosterol as well as cholesterol was 0.03 μmol/L (0.15 pmol). Total lathosterol in control human and rat serum was determined by the present method with a recovery of more than 95.8% and an RSD ( n = 5) of less than 7.3%. The present method was applied to an experiment with rats to examine the effect of lathosterol feeding. There were no significant changes in serum lathosterol or cholesterol levels in rats fed with a high-lathosterol diet for six days. Therefore, we found this method to be both simple and useful for the simultaneous determination of lathosterol and cholesterol in serum.

Impaired function and expression of P-glycoprotein in blood–brain barrier of streptozotocin-induced diabetic rats

The aim was to investigate the effect of diabetes mellitus (DM) on P-glycoprotein (P-GP) function and expression in rat blood–brain barrier (BBB). P-GP function in BBB was assessed by measuring the brain-to-plasma concentration ratios (Kp values) of rhodamine 123 (Rho123) and vincristine (VCR), two well-known P-GP substrates, in control rats and 5-week streptozotocin (STZ)-induced diabetic rats. Evans blue (EB) dye was used as a BBB integrity indicator for examining the extravasation from the blood into the brain. P-GP expression in the brain cortex was evaluated with Western blot. The uptakes of Rho123 and VCR by cultured rat brain microvessel endothelial cells (rBMECs) incubated in diabetic and control rat serum for 72 h were also used to examine P-GP function, respectively. It was found that the Kp value of Rho123 (0.022 ± 0.005 vs. 0.016 ± 0.002 ml/g brain, p = 0.033) and VCR (0.072 ± 0.028 vs. 0.023 ± 0.006 ml/g brain, p = 0.006) in diabetic rats was significantly higher than that in control rats. The uptakes of Rho123 and VCR by cultured rBMECs incubated in the diabetic rat serum were higher than that in the control rat serum, respectively. No significant difference of the EB concentration in the brain cortex was found between the diabetic rats and control rats. Electron microscope examination of the brain cortex did not show a clear damage to the endothelial cells of microvessel in diabetic rats. In addition, the protein level of P-GP in the brains of the diabetic rats examined was significantly lower than that of control rats. These results suggested that the function and expression of P-GP might be impaired in the BBB of STZ-induced diabetic rats.

Early and transient cytotoxic response of peritoneal cells from Fasciola hepatica-infected rats.

Experimental infection by F. hepatica was performed on rats. Early recruitment of the peritoneal cell population was observed and revealed transient parasite-killing activity, preceded and followed by a state of total unresponsiveness. The activation peaked at seven days post-infection (dpi) and was characterised by a massive peritoneal cell recruitment, a strong superoxide anion and nitric oxide (NO) production, that were coincident with the fasciolicide activity of these cells, as monitored by an in vitro decrease of juvenile fluke viability in a conditioned medium. The addition of L-NG-monomethyl arginine (LNMMA) to cell cultures abrogated both fasciolicide activity and NO production. Parasites started to die when NO production exceeded 25 microM and all juvenile flukes were killed by a 90 microM NO exposition (Lethal Dose 50 between 45.8 and 50.3 microM, 95% fiducial limits). However, when rat peritoneal cells were cultured in the presence of either infected or control rat serum, juvenile flukes were much more resistant to the oxidative burst, despite a massive attachment of rat peritoneal cells to the parasite tegument. These data suggest that a transient control of fasciolosis may take place in the peritoneum following the parasite intrusion but that the parasite efficiently scavenges the host cellular response to avoid destruction.

ELISA measurement of stachylysin in serum to quantify human exposures to the indoor mold Stachybotrys chartarum.

The goal of this research was to develop a measurable indicator of human exposure to Stachyborys chartarum. Antibodies were produced against the hemolytic agent stachylysin obtained from the mold S. chartarum. These antibodies were used to develop two enzyme-linked immunosorbent assay methods for the analysis of stachylysin in human and rat sera and environmental samples. Stachylysin was measured in rat pups that received nasal instillations of S. chartarum conidia but not in control rat serum. Stachylysin in the serum of five human adults exposed to S. chartarum in water-damaged environments was 371 ng/mL but none was detected in the control serum. Stachylysin was also quantified in spore, wallboard, mycelial, and dust samples. The measurement of stachylysin may be a useful indicator in assessing human exposure to S. chartarum and in determining the presence of this indoor mold.

LC/MS determination of the enaminones E139, DM5 and DM27 in rat serum

Enaminones, E139, DM5 and DM27, have been recently recognized as potential anticonvulsant compounds. The molecular masses of these enarminones were proven using ion trap Finnigan mass spectrometer. For conduction of biological studies in animals, a sensitive and selective high-performance liquid chromatography-mass spectrometry (LC/MS) was developed for the determination of the selected enaminones in rat serum. A simple protein precipitation procedure was followed for cleaning up the serum samples before analysis. LC/MS determinations were performed using an APCI probe at 430 °C. Positive ions (M+1) + were acquired in MS/MS-SRM mode at m/ z 308.1 (parent m/ z 340.2) for E139 and m/ z 262.1 (parent m/ z 294.1) for DM5. On the other hand, DM27 and E118 (internal standard) were measured in SIM mode at m/ z 236.5 and 222.5, respectively. Quantitation was based on measurement of the peak area ratio of enaminones (E139, DM5, DM27) and E118 as an internal standard. Calibration curves were linear ( r>0.9989) over the concentration range 100–1000 ng ml −1 and were free from serum interference. Precision and accuracy studies of control samples showed intra-day and inter-day %RSD <10.1 and % deviation from nominal concentrations (%DEV) from −4.3 to +10.1. Recoveries of E139, DM5 and DM27 from quality control rat serum samples using protein precipitation method were 92.3, 89.4 and 89.6%, respectively. The reported data suggest the utility of this developed method for structural elucidation and for performing pharmacokinetics studies on the selected enaminones in rats.

Diabetes Enhances Lectin-like Oxidized LDL Receptor-1 (LOX-1) Expression in the Vascular Endothelium: Possible Role of LOX-1 Ligand and AGE

Diabetes mellitus accelerating atherosclerosis was associated with the enhanced glycoxidative modification of lipoproteins. LOX-1, the endothelial oxidized LDL receptor might be involved in the pathogenesis of diabetic atherosclerosis. In this study, we examined the vascular expression of LOX-1 in streptozotocin-induced diabetic rats. We found that LOX-1 was significantly increased in diabetic rat aorta compared with nondiabetic control. Immunohistochemistry revealed that the most distinctive staining of LOX-1 was in the endothelial cells, especially in the bifurcations of artery branches from aorta. In cultured aortic endothelial cells, diabetic rat serum and advanced glycation endproducts–BSA induced LOX-1 expression, while control rat serum along with high glucose did not. Applying a competitive inhibition assay, we found that LOX-1 ligand activity was accumulated in the diabetic rat serum, mainly in VLDL/LDL fractions. In addition, VLDL/LDL prominently increased LOX-1 among all the lipoprotein fractions of diabetic rat serum. In conclusion, diabetes markedly upregulated LOX-1 expression in the aortic endothelial cells. The enhanced glycoxidative modification of lipoproteins may contribute to the underlying mechanisms.

Enzyme Immunoassay for Aflatoxin B1-Lysine Adduct and Its Validation

A simple procedure was developed for in vitro synthesis and characterization of aflatoxin B1-lysine adduct using aflatoxin B1, N-alpha-acetyl lysine and m-chloroperbenzoic acid (MCPBA). At a molar ratio of 1:16 (aflatoxin B1:N-alpha-cetyl lysine), the recovery of adduct was 62%. Analysis of the adduct by thin-layer chromatography showed a single spot (Rf = 0). Absorption spectra of the adduct showed 2 peaks at 275 and 335 nm. Liquid chromatographic (LC) analysis of the AFB1-lysine adduct showed a relative retention time of 2.1 min. Using the same epoxidation procedure, BSA-AFB1 adduct and ovalbumin-AFB1 adduct were synthesized for production of antibodies and as coating antigen, respectively. Control rat serum, spiked with AFB1-lysine adduct and subjected to LC analysis showed a retention time of 2.1 min, which is similar to that of AFB1-lysine reference standard, synthesized. Further, enzymatically hydrolyzed, control rat serum spiked with BSA-AFB1 adduct showed 2 peaks with retention times of 2.1 and 2.7 min. Based on the LC analysis, recovery of BSA-AFB1 in terms of AFB1-lysine adducts was 67 +/- 5%. The major peak (2.1 min) accounted for 72% of the adduct; the second minor peak (2.7 min) accounted for 28% of the total AFB1-lysine adducts formed. Stability studies on the AFB1-lysine adduct synthesized, indicated that it was stable for 1 month. Antibody capture assay showed an absorbance of 0.9 to 1.0 at a dilution of 1:50,000 when ovalbumin-AFB1 was used as a coating antigen. Indirect competitive ELISA showed 50% displacement (IC50) of the antibodies at a concentration of 13 ng AFB1-lysine, whereas the IC50 for AFB1 was 7 ng. The recovery of AFB1-lysine adduct spiked to control rat serum followed by enzymatic hydrolysis and immunoanalysis (indirect ELISA) was 93 +/- 6%. The enzyme immunoassay was validated by a rodent model, in which the animals were exposed to aflatoxin B1 (20 microg AFB1/kg body mass/day). The level of AFB1-lysine adduct in the rat serum was 27.3 +/- 4.37 microg/mg albumin.

Inhibition of collagen biosynthesis and increases in low molecular weight IGF-I binding proteins in the skin of fasted rats.

Insulin-like growth factor-I (IGF-I) is an important stimulator of collagen biosynthesis and prolidase activity in connective tissue cells. The disturbances in skin collagen metabolism (reflected by significant decrease in skin collagen content, collagen biosynthesis and prolidase activity) in fasted rats were accompanied by decrease in serum IGF-I level. Fasted rat serum was found to contain about 58% of IGF-I (101.6 +/- 15.4 ng/ml) as compared to control rat serum (175.7 +/- 19.8 ng/ml), while the skin of control and fasted rats contained similar concentrations of IGF-I (about 77 ng/g tissue). The insulin-like growth factor binding proteins (IGFBPs) of sera and tissue extracts (known to regulate IGF-I activity) were analysed by ligand blotting. In the serum of control rats one IGFBP band of about 46 kDa (corresponding to the acid-dissociated IGFBP-3) was detected. In the serum of fasted rats the 46 kDa IGFBP was not observed, however, an other IGFBP of about 30 kDa (corresponding to low molecular weight IGFBPs, e.g. IGFBP-1 or IGFBP-2) was found. The intensity of IGF-I binding to the 30 kDa IGFBP was much higher than that of IGFBP-3, found in control rat serum. Control and fasted rat skin contained similar IGFBPs, however their IGF-I binding abilities were much lower, compared to their serum counterparts. It was found that 46 kDa and 30 kDa proteins, observed in ligand blotting represent IGFBP-3 and IGFBP-1 or IGFBP-2. respectively as demonstrated by western immunoblot analysis. An increase in IGF-binding to 30 kDa IGFBP-1 and/or IGFBP-2 (known as an inhibitors of IGF-dependent functions) in the skin of fasted rats may explain the mechanism of reduced collagen biosynthesis and deposition in tissues during fasting.

Potential role of pyrroline 5-carboxylate in regulation of collagen biosynthesis in cultured human skin fibroblasts

Although insulin-like growth factor-I (IGF-I) is known as an important stimulator of collagen biosynthesis in collagen-producing cells, the mechanism and endpoints by which it regulate the process remain largely unknown. Serum of acutely fasted rats contained reduced amount of IGF-I (72±16 ng/ml) and showed about 75% reduced ability to stimulate collagen and DNA synthesis in confluent human skin fibroblasts in comparison to the effect of control rat serum (IGF-I, 168±19 ng/ml). An addition of IGF-I (at least 40 ng/ml) to fasted rat serum restored its mitogenic activity but could not restore its ability to stimulate collagen biosynthesis to control values during 24 h of incubation. However, when the cells were incubated in fasted rat serum supplemented with 40 ng/ml of IGF-I for 48 h, collagen biosynthesis was restored to control values. It suggests that the stimulatory role of IGF-I in collagen biosynthesis undergo indirectly. We considered pyrroline-5-carboxylate (P5C) as a candidate to play a direct role in this process. Since IGF-I and P5C are known to be decreased in serum of fasted rats it seems that the action of IGF-I on collagen biosynthesis may involve participation of P5C. We have found that serum of fasted rats (showing low level of P5C) supplemented with 1 mmol/l P5C induced collagen biosynthesis in confluent human skin fibroblasts during 24 h to control values. Supporting evidence comes from the experiment showing stimulatory action of P5C on collagen biosynthesis in fibroblasts cultured in serum-free medium. Our results postulate potential role of P5C in regulation of collagen biosynthesis and indicate participation of this molecule in the pathway of IGF-I action in this process.

Insulin-like growth factor-binding protein-3 protease activity in Snell normal and Pit-1 deficient dwarf mice.

Partial proteolysis of insulin-like growth factor-binding protein-3 (IGFBP-3) lowers its affinity for IGFs. Presumably, this leads to destabilization of the ternary IGF-IGFBP-3-acid-labile subunit complex in the circulation and an increased bioavailability of IGFs. We investigated the effect of GH on IGFBP-3 proteolysis by comparing serum from normal mice and GH-deficient dwarf mice. While normal mouse serum degraded 125I-IGFBP-3, this activity declined with age. In contrast, serum from dwarf mice displayed strong proteolytic activity at all ages tested (up to 10 weeks). In dwarf mice of 4 weeks and older, this activity could not be inhibited by EDTA and 1,10-phenanthroline, indicating the presence of a divalent cation-independent protease. Prolonged treatment with GH (4 weeks) did not decrease the overall potency of the serum to degrade IGFBP-3, but partially restored the ability of EDTA to inhibit IGFBP-3 protease activity. GH deficiency therefore appears to induce a new kind of IGFBP-3 protease. Similarly, serum from hypophysectomized rats displayed enhanced IGFBP-3 protease activity compared with control rat serum. These results suggest that a protease induced under conditions of severe GH deficiency may contribute to making IGFs optimally available to the tissues.

Serum DNA polymerase β as an indicator for fatal liver injury of rat induced by d-galactosamine hydrochloride and lipopolysaccharide

DNA polymerase β (pol β) is a nuclear enzyme that is tightly bound to chromatin. Release of the pol β activity into serum, therefore, may indicate the occurrence of massive destruction of cell nuclei in organs or tissues. In the present study, we made a liver injury model rat by the intraperitoneal injection of d-galactosamine hydrochloride (GalN, 500 mg/kg) and lipopolysaccharide (LPS, 100 μg/kg). Serum from the GalN/LPS-treated rats showed a high level of pol β activity up to 118 pmol/0.5 μl serum (4700 cpm) at 12 h after the treatment, while the control rat serum showed the back ground level (3.8 pmol/0.5 μl, 150±70 cpm). The serum pol β activity was sensitive to inhibition by 2′,3′-dideoxyTTP and by an anti-rat pol β antibody. Among 30 rats treated with GalN/LPS, 10 rats died within 120 h (dead group). Serum pol β activity in the dead group was as high as 23.0±19.5 pmol/0.5 μl (925±778 cpm) at 10 h after the treatment, while in alive group ( n=20), it was 3.7±3.2 pmol. Levels of the serum pol β activity correlated well with the prognosis of GalN/LPS-treated rats based on an analysis of the receiver-operator characteristic curves.