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Objectives Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has considerable applicative potential for both qualitative and quantitative analyses of elemental spatial distribution and concentration. It provides high resolutions at pg-level detection limits. These qualities make it very useful for analyzing biological samples. The present study responds to the growing demand for adequate analytical methods which would allow to assess the distribution of nanostructured molybdenum(IV) disulfide (MoS 2 ) in organs. It was also motivated by an apparent lack of literature on the biological effects of MoS 2 in living organisms. The study was aimed at using LA-ICP-MS for comparing micro- and nanosized MoS 2 ditribution in selected rat tissue samples (lung, liver, brain and spleen tissues) after the intratracheal instillation (7 administrations) of MoS 2 nano- and microparticles vs. controls. Material and Methods The experimental study, approved by the Ethics Committee for Animal Experiments was performed using albino Wistar rats. This was performed at 2-week intervals at a dose of 5 mg/kg b.w., followed by an analysis after 90 days of exposure. The MoS 2 levels in control tissues were determined with the laser ablation system at optimized operating conditions. The parameter optimization process for the LA system was conducted using The National Institute of Standards and Technology (NIST) glass standard reference materials. Results Instrument parameters were optimized. The study found that molybdenum (Mo) levels in the lungs of microparticle-exposed rats were higher compared to nanoparticle-exposed rats. The opposite results were found for liver and spleen tissues. Brain Mo concentrations were below the detection limit. Conclusions The LA-ICP-MS technique may be used as an important tool for visualizing the distribution of Mo on the surface of soft samples through quantitative and qualitative elemental mapping. Int J Occup Med Environ Health. 2024;37(1):18–33

Objectives: Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has considerable applicative potential for both qualitative and quantitative analyses of elemental spatial distribution and concentration. It provides high resolutions at pg-level detection limits. These qualities make it very useful for analyzing biological samples. The present study responds to the growing demand for adequate analytical methods which would allow to assess the distribution of nanostructured molybdenum(IV) disulfide (MoS2) in organs. It was also motivated by an apparent lack of literature on the biological effects of MoS2 in living organisms. The study was aimed at using LA-ICP-MS for comparing micro- and nanosized MoS2 ditribution in selected rat tissue samples (lung, liver, brain and spleen tissues) after the intratracheal instillation (7 administrations) of MoS2 nano- and microparticles vs. controls. Material and Methods: The experimental study, approved by the Ethics Committee for Animal Experiments was performed using albino Wistar rats. This was performed at 2-week intervals at a dose of 5 mg/kg b. w., followed by an analysis after 90 days of exposure. The MoS2 levels in control tissues were determined with the laser ablation system at optimized operating conditions. The parameter optimization process for the LA system was conducted using The National Institute of Standards and Technology (NIST) glass standard reference materials. Results: Instrument parameters were optimized. The study found that molybdenum (Mo) levels in the lungs of microparticle-exposed rats were higher compared to nanoparticle-exposed rats. The opposite results were found for liver and spleen tissues. Brain Mo concentrations were below the detection limit. Conclusions: The LA-ICP-MS technique may be used as an important tool for visualizing the distribution of Mo on the surface of soft samples through quantitative and qualitative elemental mapping. [ABSTRACT FROM AUTHOR]

Dolutegravir (DTG) has been introduced into first line combination antiretroviral therapy (ART) for HIV/AIDS. Penetration of ART into HIV reservoirs is essential to prevent continuous replication of HIV. However, accumulation of DTG in HIV reservoirs could be contributing to the adverse effects reported. We have developed and applied liquid chromatography tandem mass spectrometry (LC-MS/MS) methods to quantify DTG in wistar rat biological matrices following chronic DTG administration. DTG was detected using a Shimadzu 8040 triple quadrupole-mass spectrometer. The methods developed were in the concentration ranges of 17.5–8000 ng/mL for plasma and 15.5–16 680 ng/g for tissue matrices. Mean plasma DTG concentrations in the current study closely corresponded to plasma DTG levels reported in humans after chronic treatment. Plasma and tissue DTG concentrations were generally higher in females compared to male wistar rats, but these differences were nullified after correcting for body and organ size. Plasma DTG levels correlated with tissue DTG concentrations in the liver and gastrocnemius muscle tissue. Data suggest that body size – rather than sex – may be a major risk factor determining adverse outcomes of patients on the current DTG dosing strategy which does not account for differences in body mass. Furthermore, plasma DTG was not correlated with adipose tissue DTG concentration. This suggests that adipose may be a primary site for longer term inflammatory dysregulation and adverse outcome following DTG treatment.

Exposure to paraoxon (POX) and leptin (LP) could cause an imbalance between oxidants and antioxidants in an organism, which can be prevented by introduction of exogenous antioxidants such as N-acetylcysteine (NAC). The aim of this study was to evaluate synergic or additive effects of administration of exogenous LP plus POX on the antioxidant status, as well as the prophylactic and therapeutic roles of NAC in various rat tissues. Fifty-four male Wistar rats were divided into nine groups treated with different compounds: Control (no treatment), POX (0.7 mg/kg), NAC (160 mg/kg), LP (1 mg/kg), POX+LP, NAC-POX, POX-NAC, NAC-POX+LP, and POX+LP-NAC. In the last five groups, only the order of administered compounds differed. After 24 h, plasma and tissues were sampled and examined. The results showed that administration of POX plus LP significantly increased biochemical indices in plasma and antioxidant enzymes activities and decreased glutathione content in the liver, erythrocytes, brain, kidney, and heart. In addition, cholinesterase and paraoxonase 1 activities in the POX+LP-treated group were decreased and malondialdehyde level was increased in the liver, erythrocytes, and brain. However, administration of NAC rectified induced changes although not to the same extent. Our study suggests that POX or LP administration engage the oxidative stress system per se; however, their combination did not produce significantly greater effects. Moreover, both prophylactic and therapeutic treatments of rats with NAC supported the antioxidant defense against oxidative damage in tissues, most probably through both its free radical scavenging ability and maintaining intracellular GSH levels. It can therefore be suggested that NAC has particularly protective effects against POX or/and LP toxicity. [ABSTRACT FROM AUTHOR]

Lithium (Li) represents a first choice mood stabilizer for bipolar disorder (BD). Despite extensive clinical use, questions regarding its mechanism of action and pathological mechanism of renal function impairment by Li remain open. The present study aimed to improve our knowledge in this area paying special attention to the relationship between the length of Li action, lipid peroxidation (LP), and Na+/K+-ATPase properties. The effects of therapeutic Li doses, administered daily to male Wistar rats for 1 (acute), 7 (short term) and 28 days (chronic), were studied. For this purpose, Na+/K+-ATPase activity measurements, [3H]ouabain binding and immunoblot analysis of α-Na+/K+-ATPase were performed. Li-induced LP was evaluated by determining the malondialdehyde concentration by HPLC. Sleep deprivation (SD) was used as an experimental approach to model the manic phase of BD. Results obtained from the kidney were compared to those obtained from erythrocytes and different brain regions in the same tested animals. Whereas treatment with therapeutic Li concentration did not bring any LP damage nor significant changes of Na+/K+-ATPase expression and [3H]ouabain binding in the kidney, it conferred strong protection against this type of damage in the forebrain cortex. Importantly, the observed changes in erythrocytes indicated changes in forebrain cortices. Thus, different resistance to SD-induced changes of LP and Na+/K+-ATPase was detected in the kidney, erythrocytes and the brain of Li-treated rats. Our study revealed the tissue-specific protective properties of Li against LP and Na+/K+-ATPase regulation. [ABSTRACT FROM AUTHOR]

Glutathione S-transferases are multifunctional enzymes for the cellular defense against xenobiotics and provide protection for organism. In this study, the inhibition effects of some antibiotics were investigated against GST obtained from albino-rats kidney, liver, and heart tissues. Ninety-six albino-rats were randomly divided into 16 groups (n:6). The first four groups were control groups that were administrated blank enjection and decapitated at 1–7 h. The other groups were administrated the antibiotics. In all tissues, GST activity was increased in antibiotics groups at 1st and 3rd hours compared to control groups, while it began to fall at 5th and 7th hours (p <.05). In kidney tissues, it was lower than the same control group the cefuroxime and cefoperazone groups at 7th hours (p <.05). In addition, almost all antibiotic groups of kidney tissues had higher GST activity at all hours than those of control groups, but it was higher only at 5th hours in heart tissues (p <.05). [ABSTRACT FROM AUTHOR]

Antioxidant enzymes play an important role in body defense and free radical removal. Cephalosporins are β-lactam antibiotics. In this work, the effects of cefazolin, cefuroxime and cefoperazone which are cephalosporins on some selected antioxidant enzyme and levels of malondialdehyde (MDA) as lipid peroxidation product were investigated in kidney, liver, and brain tissues of albino female rats. Ninety-six albino rats were randomly divided into 16 groups of equal number (n = 6). 50 mg/kg cefazolin, 25 mg/kg cefuroxime, and 100 mg/kg cefoperazone were injected intraperitoneally to the groups (5th–8th and 9th–12th, and 13th–16th groups), respectively. The changes in glutathione reductase (GR), glutathione S-transferase (GST), superoxide dismutase (SOD), peroxidase (POD), and glutathione peroxidase (GSH-Px) levels were studied in each time point group and a time-dependent manner (at the 1st, 3rd, 5th and 7th hour). In addition, MDA levels were examined in all the tissues. The drugs evaluated in this study had different effects on the same enzyme in different tissues depending on time. MDA levels especially in cefazolin and cefoperazone experiments were lower in all the tissues; however, MDA levels were higher in brain and kidney tissues in the cefuroxime groups in a time-dependent manner (p < 0.05). These results revealed the complex effects of the tested drugs on different tissues at different time points. Therefore, the dose and use of these drugs should be adjusted correctly. [ABSTRACT FROM AUTHOR]

In our study, it was aimed to investigate the effects of cefazolin, cefuroxime, and cefoperazon injected to rats on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzyme activities in the heart, brain, eye, liver, and kidney tissues of rats. Liver AChE activity at the 1st and 3rd hours of cefuroxime groups was higher than the control group at the same time (p <.05). The AChE activity of the heart tissue decreased in the cefazolin group compared to the control group at the same hour, whereas it increased in the cefuroxime group (p <.05). AChE activities of kidney tissue of cefazolin and cefuroxime groups were lower than those of the same control group on the 3rd and started to increase on the next hours (p <.05). BChE activity is measured in tissues increased within the first three hours and decreased significantly within the first hour in the cefoperazone group (p <.05). [ABSTRACT FROM AUTHOR]

As a result of thermal treatments of tomatoes, a significant lycopene degradation is produced, being modulated this effect by the moisture content. It is of great interest, as a consequence, to know the effect of thermal processes on nutritional properties of tomatoes. The information obtained may be a valuable tool to optimize the conditions of thermal processing, mainly temperature and water content, to minimize lycopene degradation. In the present study, we compared the efficiency of three types of processed tomatoes, i.e., Cold Break, Oleoresin, and Powder, as protection agents against oxidative stress induced by carbon tetrachloride (CCl4) in the liver, kidney, heart, lung, and brain tissues of rats. To check the antioxidant properties of these processed, rats were pre-treated with Cold Break, Oleoresin, and Powder processed tomatoes, and then intoxicated with CCl4. Activities of serum glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), and lactate dehydrogenase (LDH) were measured to check the hepatic damage in rats. With the aim of evaluating the protective efficiency of processed tomatoes in rat tissues, we measured the concentration of chemical stress oxidative markers H2O2, malonyldialdehyde (MDA) and reduced glutathione (GSH), and the activities of catalase (CAT), superoxide dismutase (SOD), glutathione-S-transferase (GST), glutathione reductase (GRase), and glutathione peroxidase (GPx). Our results clearly showed that, although the efficiencies of the three types of processed tomatoes were not the same and that the effect in each tissue was slightly different, the Oleoresin preparation showed a protective effect against organ oxidative stress that was slightly higher than tomato Powder or Cold Break products. [ABSTRACT FROM AUTHOR]

Glutaryl-CoA dehydrogenase (GCDH) is a mitochondrial enzyme that is involved in the degradation of tryptophan, lysine and hydroxylysine. Deficient enzyme activity leads to glutaric aciduria type-I (GA-I). This neurometabolic disease usually manifests with acute encephalopathic crises and striatal neuronal death in early childhood leading to an irreversible dystonic-dyskinetic movement disorder. Fronto-temporal atrophy and white matter changes are already present in the pre-symptomatic period. No detailed information on GCDH expression during embryonic development and in adulthood was available so far. Using immunofluorescence microscopy and cell-type-specific markers to localize GCDH in different tissues, we describe the differential cellular localization of GCDH in adult rat brain and peripheral organs as well as its spatiotemporal expression pattern. During embryonic development GCDH was predominantly expressed in neurons of the central and peripheral nervous system. Significant expression levels were found in epithelial cells (skin, intestinal and nasal mucosa) of rat embryos at different developmental stages. Besides the expected strong expression in liver, GCDH was found to be significantly expressed in neurons of different brain regions, renal proximal tubules, intestinal mucosa and peripheral nerves of adult rats. GCDH was found widely expressed in embryonic and adult rat tissues. In rat embryos GCDH is predominantly expressed in brain implying an important role for brain development. Interestingly, GCDH was found to be significantly expressed in different other organs (e.g. kidney, gut) in adult rats probably explaining the evolving phenotype in GA-I patients. [ABSTRACT FROM AUTHOR]

Biodegradable magnesium implants are under investigation because of their promising properties as medical devices. For enhancing the mechanical properties and the degradation resistance, rare earth elements are often used as alloying elements. In this study Mg10Gd pins were implanted into Sprague–Dawley® rats. The pin volume loss and a possible accumulation of magnesium and gadolinium in the rats’ organs and blood were investigated in a long-term study over 36 weeks. The results showed that Mg10Gd is a fast disintegrating material. Already 12 weeks after implantation the alloy is fragmented to smaller particles, which can be found within the intramedullary cavity and the cortical bones. They disturbed the bone remodeling until the end of the study. The results concerning the elements’ distribution in the animals’ bodies were even more striking, since an accumulation of gadolinium could be observed in the investigated organs over the whole time span. The most affected tissue was the spleen, with up to 3240 μg Gd/kg wet mass, followed by the lung, liver and kidney (up to 1040, 685 and 207 μg Gd/kg). In the brain, muscle and heart, the gadolinium concentrations were much smaller (less than 20 μg/kg), but an accumulation could still be detected. Interestingly, blood serum samples showed no accumulation of magnesium and gadolinium. This is the first time that an accumulation of gadolinium in animal organs was observed after the application of a gadolinium-containing degradable magnesium implant. These findings demonstrate the importance of future investigations concerning the distribution of the constituents of new biodegradable materials in the body, to ensure the patients’ safety. Statement of Significance In the last years, biodegradable Mg alloys are under investigation due to their promising properties as orthopaedic devices used for bone fracture stabilization. Gadolinium as Rare Earth Element enhances the mechanical properties of Mg-Gd alloys but its toxicity in humans is still questionable. Up to now, there is no study investigating the elements’ metabolism of a REE-containing Magnesium alloy in an animal model. In this study, we examined the gadolinium distribution and accumulation in rat organs during the degradation of Mg10Gd. Our findings showed that Gd is accumulating in the animal organs, especially in spleen, liver and kidney. This study is of crucial benefit regarding a safe application of REE-containing Magnesium alloys in humans. [ABSTRACT FROM AUTHOR]

To evaluate the time effect on the distribution of zinc oxide nanoparticles (ZnO NPs) in tissues from rats and mice, a search on the PubMed, Embase, SpringerLink, Scopus, Science Direct, Cochrane, CNKI, Wanfang, and vip databases up to September 2014 was performed, followed by screening, data extraction, and quality assessment. Thirteen studies were included. At 24 h, Zn content was mainly distributed in the liver, kidney, and lung. At ≥7 days, Zn content was mainly distributed in the liver, kidney, lung, and brain. ZnO NPs are readily deposited in tissues. Furthermore, as time increases, Zn content decreases in the liver and kidney, but increases in the brain. [ABSTRACT FROM AUTHOR]

Most studies have focused on the biodistribution of titanium(IV) oxide as nanoparticles or crystals in organism. But several reports suggested that titanium is released from implant in ionic form. Therefore, gaining insight into toxicokinetics of Ti ions will give valuable information, which may be useful when assessing the health risks of long-term exposure to titanium alloy implants in patients. A micro synchrotron radiation-induced X-ray fluorescence (µ-SRXRF) was utilized to investigate the titanium distribution in the liver, spleen and kidneys of rats following single intravenous or 30-days oral administration of metal (6 mg Ti/b.w.) in ionic form. Titanium was mainly retained in kidneys after both intravenous and oral dosing, and also its compartmentalization in this organ was observed. Titanium in the liver was non-uniformly distributed-metal accumulated in single aggregates, and some of them were also enriched in calcium. Correlation analysis showed that metal did not displace essential elements, and in liver titanium strongly correlated with calcium. Two-dimensional maps of Ti distribution show that the location of the element is characteristic for the route of administration and time of exposure. We demonstrated that µ-SRXRF can provide information on the distribution of titanium in internal structures of whole organs, which helps in enhancing our understanding of the mechanism of ionic titanium accumulation in the body. This is significant due to the popularity of titanium implants and the potential release of metal ions from them to the organism. [ABSTRACT FROM AUTHOR]

The tripeptide Hip-His-Leu was used to standardize a fluorimetric method to measure tissue angiotensin-converting enzyme (ACE) activity in rats. The fluorescence of the o-phthaldialdehyde-His-Leu adduct was compared in the presence and absence of the homogenate (25 µl) to determine whether the homogenate from different tissues interfered with the fluorimetric determination of the His-Leu product. Only homogenates from lung and renal medulla and cortex showed significantly altered fluorescence intensity. To overcome this problem, the homogenate from these tissues were diluted 10 times with assay buffer. The specificity of the assay was demonstrated by the inhibition of ACE activity with 3 µM enalaprilat (MK-422). There was a linear relationship between product formation and incubation time for up to 90 min for homogenates of renal cortex and medulla and liver, for up to 60 min for ventricles and adrenals and for up to 30 min for the aorta, lung and atrium homogenates. In addition, there was a linear relationship between product formation and the amount of protein in the homogenates within the following range: lung, 30-600 µg; renal cortex and medulla, 40-400 µg; atrium and ventricles, 20-200 µg; adrenal, 20-100 µg; aorta, 5-100 µg; liver, 5-25 µg. No peptidase activity against the His-Leu product (31 nmol), assayed in borate buffer (BB), was detected in the different homogenates except the liver homogenate, which was inhibited by 0.1 mM r-chloromercuribenzoic acid. ACE activity in BB was higher than in phosphate buffer (PB) due, at least in part, to a greater hydrolysis of the His-Leu product in PB. ACE activity of lung increased 20% when BB plus Triton was used. Enzyme activity was stable when the homogenates were stored at -20o or -70oC for at least 30 days. These results indicate a condition whereby ACE activity can be easily and efficiently assayed in rat tissue samples homogenized in BB using a fluorimetric method with Hip-His-Leu as a substrate.

Tandem mass spectrometry (MS/MS) has been used for the analysis of plasmalogen (Pls), a physiologically important class of vinyl ether-linked phospholipid. However, MS/MS generally causes little fragmentation of Pls, especially choline Pls (PC-Pls). Previous MS/MS studies reported an increased formation of product ions of PC-Pls (and also ethanolamine Pls (PE-Pls)) in the presence of ‘alkali metals.’ Therefore, use of alkali metals considerably leads to the development of a method for analysis of both PC- and PE-Pls. In this study, this notion was evaluated using quadrupole-time-of-flight MS/MS and liquid chromatography (LC) coupled with MS/MS. Results from MS/MS confirmed that alkali metals (e.g., sodium) produced significant fragmentation of PC-Pls and PE-Pls. A number of structure-diagnostic product ions exhibiting high intensities were observed under optimized MS/MS conditions using alkali metals. Moreover, the ability to selectively and sensitively identify PC-Pls and PE-Pls at the molecular species level in biological samples (rat brain and heart) was demonstrated using LC-MS/MS. Therefore, the herein developed method appears to be a powerful tool for analyzing Pls and may provide a better understanding of their physiological roles in vivo. [ABSTRACT FROM AUTHOR]

The quantitative analysis of taurine and edaravone in biological sample is critical in pharmaceutical studies. Although each of them can be individually analyzed by different approaches, concurrent quantification is still a highly challenging task with respect to their great polarity variation and the complex composition of tissue sample. In the present study, to simultaneously determine taurine and edaravone in rat tissue, the sample preparation and chromatographic separation conditions were evaluated and discussed in detail. As for the sample preparation, four kinds of solvent and the volume ratio of the optimal solvent to biological sample were both tested and evaluated based on the chromatographic profile, extraction recovery, and matrix effect (ME). The chromatographic separation was performed in a reverse phase (RP) and two hydrophilic interaction liquid chromatography (HILIC) modes, and the corresponding separation efficiencies were assessed using chromatographic parameters like half-width (W 1/2 ), tailing factor (f t), theoretical plates number (N), and ME. Furthermore, adopted composition of two mobile phase systems and the concentrations of the additives in the optimum buffer system were also investigated on an Atlantis HILIC silica column according to the resultant chromatographic profiles and peak areas of the analytes. The optimal results were obtained when the biological samples were deproteined by 4-fold volume of methanol/acetonitrile (1:3, v/v) and separated on a HILIC column with a gradient elution of acetonitrile/water containing 0.2 % formic acid and 10 mM ammonium formate. The proposed approach was validated and successfully applied to the parallel determination of the tissue distribution of edaravone and taurine in rat tissues. [ABSTRACT FROM AUTHOR]

A selective, sensitive and high performance liquid chromatography-tandem mass spectrometry (LC–(ESI)MS/MS) method has been developed and validated for the quantification of the potent antitubercular drug candidate, PA-824, in rat plasma, lung and brain tissues. Sample clean-up involved protein precipitation and solid-phase extraction. Chromatographic separation was performed on YMC Triart C 18 column (150 mm × 3.0 mm, 3.0 μm). The method was validated over the concentration range of 75–1500 ng/mL for plasma, 50–1200 ng/g for lungs and 100–1500 ng/g for brain tissue. Evaluation of the pharmacokinetic properties of PA-824 utilized Sprague Dawley rats with a dosage of 20 mg/kg at various time points. The new method was applied successfully for the determination of PA-824 with liquid desorption followed by liquid chromatography with ultra-high resolution quadrupole time-of-flight mass spectrometry in the different biological samples. [ABSTRACT FROM AUTHOR]

Overexpression of P-glycoprotein leads to tumor multidrug resistance (MDR). HZ08, a novel tetrahydro-isoquinoline derivate, was discovered to inhibit the MDR in the cancer cell lines of MCF-7/ADM, K562/ADM and KBV in our previous studies. A rapid and sensitive ultra-performance liquid chromatography–tandem mass spectrometric method (UPLC–MS/MS) was developed and validated for determination of HZ08 in rat plasma and tissues after intravenous administration of HZ08 liposome injection at different doses. The analytes were extracted from plasma and tissues using protein precipitation by acetonitrile with clotrimazole as internal standard. The chromatographic separation was performed on a Thermo BDS HYPERSIL C18 column (100 mm × 4.6 mm, 2.4 μm) at a flow rate of 0.7 ml/min using 0.2% ammonium acetate solution (containing 0.1% formic acid) and methanol as mobile phase. The total run time was 4 min. The tandem mass detection was applied with electrospray ionization in positive ion selected reaction monitoring mode. The ion transitions monitored were m / z 523.5 to 342.3 for HZ08 and 277.1 to 165.1 for the internal standard, respectively. The calibration curves obtained were linear in different matrices, and the lower limit of quantification (LLOQ) achieved was 1 ng/ml for rat plasma and 0.25 ng/ml for rat tissues, respectively. The RSDs for intra- and inter-day precision were less than 15%. Extraction recovery, matrix effect and stability were satisfactory in rat plasma and tissues. The developed method was successfully applied to a pharmacokinetic study of HZ08 liposome injection following intravenous administration of 1, 3, 10 mg/kg to Sprague-Dawley rats. The data profiles revealed that HZ08 had linear pharmacokinetic properties at the tested doses, and was rapidly distributed into the systemic circulation with wide distribution throughout the body followed by a rapid elimination phase. The major distribution tissues of HZ08 in rats were lung, spleen and liver. These results provided constructive contribution to support the clinical evaluation. [ABSTRACT FROM AUTHOR]

In spite of lutein and its isomer zeaxanthin being richly available in natural sources, the role of these components on reduction of age-related macular degeneration, cancer, and cardiovascular disorders suggested that an update of the analytical procedure is required to determine the oxidative products and to understand their nutritional significance. In the present study, we have standardized and developed an improved method to obtain characteristic ions of lutein, zeaxanthin, and its major oxidative products in vivo (rats) using LC-MS (APCI). In addition, lutein and zeaxanthin isomer were separated on a C30 column with shorter run time with high resolution and calibrated on the basis of picomolar concentration on HPLC (DAD), with the lower detection limit of 0.125 for lutein and 0.128 pmol for zeaxanthin. Characteristic mass spectral ion for lutein is m/ z 568.7 [M] and 551.5 [M + H-HO] and for zeaxanthin isomer is m/ z 568.8 [M], 569.8 [M + H]. Further, optimized conditions produced structurally characteristic fragmented ions under standardized MS (APCI) conditions. Total ionic chromatogram together with fine UV-Visible and mass spectra were used to differentiate lutein isomers and its oxidative products, such as 523 [M+ H-3CH], 479 [M + H-6CH], 551 [M + H-HO], 276.43 [M-CHO], di-epoxides and 3′-oxolutein. The APCI mass spectral characteristics of major oxidative products of lutein in adult rat tissues are reported here for the first time, to our knowledge. These findings could provide new insights into lutein bioavailability and bioconversions with respect to health benefits. [ABSTRACT FROM AUTHOR]

Highlights: [•] The homogenized rat tissues are studied by Attenuated Total Reflectance FTIR spectroscopy. [•] Biochemical content of tissues such as heart, brain, liver, lung, intestine, and kidney is analysed. [•] Similarities and differences in spectral profile of each tissue are discussed in detail. [•] ATR information is compared with those obtained for tissue sections from FTIR imaging. [Copyright &y& Elsevier]

We have investigated how diabetes affects the expression of adropin (ADR) in rat brain, cerebellum, kidneys, heart, liver, and pancreas tissues. The rats in the diabetic group were administered an intraperitoneal (i.p.) injection of a single dose of 60 mg/kg streptozotocin (STZ) dissolved in a 0.1 M phosphate-citrate buffer (pH 4.5). The rats were maintained in standard laboratory conditions in a temperature between 21 and 23 °C and a relative humidity of 70 %, under a 12-h light/dark cycle. The animals were fed a standard commercial pellet diet. After 10 weeks, the animals were sacrified. ADR concentrations in the serum and tissue supernatants were measured by ELISA, and immunohistochemical staining was used to follow the expression of the hormones in the brain, cerebellum, kidneys, heart, liver, and pancreas tissues. The quantities were then compared. Increased ADR immunoreaction was seen in the brain, cerebellum, kidneys, heart, liver, and pancreas in the diabetes-induced rats compared to control subjects. ADR was detected in the brain (vascular area, pia mater, neuroglial cell, and neurons), cerebellum (neuroglial cells, Purkinje cells, vascular areas, and granular layer), kidneys (glomerulus, peritubular interstitial cells, and peritubular capillary endothelial cells), heart (endocardium, myocardium, and epicardium), liver (sinusoidal cells), and pancreas (serous acini). Its concentrations (based on mg/wet weight tissues) in these tissues were measured by using ELISA showed that the levels of ADR were higher in the diabetic rats compared to the control rats. Tissue ADR levels based on mg/wet weight tissues were as follows: Pancreas > liver > kidney > heart > brain > cerebellar tissues. Evidence is presented that shows ADR is expressed in various tissues in the rats and its levels increased in STZ-induced diabetes; however, this effect on the pathophysiology of the disorder remains to be understood. [ABSTRACT FROM AUTHOR]

Abstract: In vitro metabolism of DEHP by subcellular fractions of human brain, intestine, kidney, liver, lung, skin, testis, rat liver and recombinant CYP isoforms of human and rat was investigated using LC–MS/MS. DEHP was rapidly hydrolyzed to mono(2-ethylhexyl) phthalate (MEHP) in 12 microsomal/cytosolic fractions of selected 7 human organs and rat liver but not in microsomal fractions of human brain and human female skin. MEHP was metabolized to CYP-mediated oxidative and dealkylated metabolites in human and rat liver and at a lower rate in human intestine. Measurable amounts of mono(2-ethyl-5-hydroxyhexyl) phthalate (5-OH MEHP), mono(2-ethyl-5-oxohexyl) phthalate (5-Oxo MEHP), mono(2-ethyl-5-carboxypentyl) phthalate (5-carboxy MEPP), mono(2-carboxymethyl-hexyl) phthalate (2-carboxy MMHP) and phthalic acid (PA) were formed by human liver fractions. Human CYP2C9∗1, CYP2C19 and rat CYP2C6 were the major CYP isoforms producing 5-OH MEHP and 5-Oxo MEHP metabolites; however, only human CYP2C9∗1 and 2C9∗2 produced 5-carboxy MEPP from MEHP. Additionally, human CYP3A4 and rat CYP3A2 were the primary enzymes for PA production via heteroatom dealkylation of MEHP. Percent total normalized rates (%TNR) by CYP2C9∗1 in human liver microsomes (HLM) were 94%, 98% and 100%, respectively, for 5-OH MEHP, 5-Oxo MEHP, 5-carboxy MEPP, and 76% for PA production by CYP3A4. [Copyright &y& Elsevier]

The effects of long-term selenium supplementation on the selenium body status were investigated in humans and rats. Selenium was determined in human muscle biopsies and monitor materials and in rat tissues by neutron activation analysis. The results showed that the body selenium load is raised by additional supply of selenomethionine or selenomethionine-containing yeast but not proportionally to the intake. The surplus selenium can serve as an endogenous source to maintain the selenoprotein levels during insufficient supply. Highly significant correlations between the muscle selenium concentrations and those in blood, blood fractions, hair and nails indicate that the selenium status can be assessed by analysis of these monitor materials. [ABSTRACT FROM AUTHOR]

In this study, the aim was to investigate possible effects of Electromagnetic Radiation (EMR) use on oxidant and antioxidant status in erythrocytes and kidney, heart, liver, and ovary tissues from rats, and possible protective role of vitamin C. For this aim, 40 Wistar albino female rats were used throughout the study. The treatment group was exposed to EMR in a frequency of 900 MHz, the EMR plus vitamin C group was exposed to the same EMR frequency and given vitamin C (250 mg/kg/day) orally for 4 weeks. There were 10 animals in each group including control and vitamin C groups. At the end of the study period, blood samples were obtained from the animals to get erythrocyte sediments. Then the animals were sacrificed and heart, kidney, liver, and ovary tissues were removed. Malondialdehyde (MDA) levels and superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), xanthine oxidase (XO), and adenosine deaminase (ADA) enzyme activities were measured in the tissues and erythrocytes. It was observed that MDA level, XO, and GSH-Px activities significantly increased in the EMR group as compared with those of the control group in the erythrocytes. In the kidney tissues, it was found that MDA level and CAT activity significantly increased, whereas XO and ADA activities decreased in the cellular phone group as compared with those of the control group. However, in the heart tissues it was observed that MDA level, ADA, and XO activities significantly decreased in the cellular phone group as compared with those of the control group. The results suggest that EMR at the frequency generated by a cell phone causes oxidative stress and peroxidation in the erythrocytes and kidney tissues from rats. In the erythrocytes, vitamin C seems to make partial protection against the oxidant stress. [ABSTRACT FROM AUTHOR]

Abstract: This study evaluates the interaction of selenium (Se) and mercury (Hg) in the accumulations and oxidative stress of rat tissues. Rats were divided into five groups including one control (n=9) and four treated groups including M-Hg (n=9), L-Hg+Se (n=11), M-Hg+Se (n=10), and H-Hg+Se (n=10) group. Treated groups of rats were instilled with different amounts of mercuric chloride (HgCl2) and dl-selenomethionine (SeMet) by gavage since pregnancy of their mothers. Atomic fluorescence spectroscopy (AFS) was applied for mercury and selenium quantification. Glutathione (GSH), malondialdehyde (MDA), and total superoxide dismutase (SOD) activity of tissues were detected using biochemical methods. Results showed that Hg was deposited mainly in kidney. Se could decrease Hg content in kidney but increase it in blood and liver. Hg decreased GSH and SOD and increased MDA levels in most detected tissues, while Se took on a counteraction effect in same tissues. This study suggests that interactions of Se and Hg affect their accumulation and Se may antagonize Hg-induced inhibition on organic activities. [Copyright &y& Elsevier]

A simple HPLC method has been developed and validated for the determination of lovastatin in rat tissues. Samples were prepared by a simple protein precipitation. Separation was carried out on a C18 column with a mobile phase of acetonitrile:0.05 M ammonium acetate, a flow rate of 1.0 mL min−1 and with detection at 238 nm. There was no interference from endogenous tissue compounds. The calibration curve was linear from 0.0175 to 7.0 μg mL−1 with a limit of detection of 0.006 μg mL−1. The method was used to measure the concentration of lovastatin in rat tissue after a single oral dose. The highest level was observed in the liver, then in kidney, heart and spleen; the lowest level was found in the brain. These results suggest that lovastatin distributes rapidly into all tissues and particularly the liver. [ABSTRACT FROM AUTHOR]

Abstract: Effects of nicotine, and nicotine+vitamin E on glutathione reductase (Glutathione: NADP+ oxidoreductase, EC 1.8.1.7) activity in the muscle, heart, lungs, testicles, kidney, stomach, brain and liver tissues were investigated in vivo and also in vitro. The groups were: nicotine [0.5 mg/kg/day, intraperitoneal (i.p.)]; nicotine+vitamin E [75 mg/kg/day, intragastric (i.g.)]; and control group (receiving only vehicles). There were eight rats per group and supplementation period was 3 weeks. The results showed that nicotine (0.5 mg/kg, i.p.) inhibited glutathione reductase activity significantly in the liver, lungs, heart, stomach, kidney, and testicles by ∼61.5%, ∼65%, ∼70.5%, ∼72.5%, ∼64% and ∼71.5%, respectively, while it had activated glutathione reductase activity in the brain by ∼11.8%, and had no effect on the muscle glutathione reductase activity. Vitamin E supplementation prevented this nicotine-induced decrease in glutathione reductase activity in liver, lungs, heart, stomach, and kidney. However, it did not prevent this nicotine-induced decrease in testicles. In vitro studies were also carried out to elucidate the effects of nicotine and vitamin E on glutathione reductase activity. In vitro results correlated well with in vivo experimental results in liver, lungs, heart, stomach, and testicular tissues. These results show that vitamin E administration generally restores the inactivation of glutathione reductase activity due to nicotine administration in various rat tissues in vivo, and also in vitro. [Copyright &y& Elsevier]

A neutral cysteine protease, bleomycin hydrolase (BH), was found to be present in the range 3.7–131.1 ng per mg of rat tissues by enzyme-lined immunosorbent assay (ELISA). Newborn rat skin contained the highest amount of BH, and relatively high levels of BH were detected in the kidney and liver of 6-week-old male rats. The tissue distribution of BH in female rats was similar to that in male rats. Moreover, BH was detected in the extracts of erythrocytes and leukocyte-rich cells as well as in those of rat hemo-lymphocytic lineage cell lines by Western blotting. The BH level was increased at 6 weeks after birth and then slightly decreased. By immunohistochemistry, BH was localized as granular staining in the distal and proximal tubular cells of the kidney, and it was also detected in hepatocytes of the liver, in the red pulpy region of the spleen and in neurons of the brain. An immunoelectron microscopic study showed that BH-immunoreactivity was essentially located in the cytoplasm and at the outer membrane of the rough endoplasmic reticulum of epithelial cells of the kidney, as well as in that of hepatocytes of the liver. These results suggest that BH may play ubiquitous and unique roles in rat tissues. [ABSTRACT FROM PUBLISHER]

The main objective of this study was to investigate the changes in free and protein-bound SH contents in methyl parathion-exposed rat tissues. The free and protein-bound SH levels are usually affected and depleted by oxidative stress-inducing agents. Results would indicate if methyl parathion toxicity partly results from depletion of sulfhydryl content of tissues. Six-week-old male Wistar albino rats were used in this study. Following exposure to methyl parathion for 3 months, the liver, the brain, and the kidney tissues were removed from the rats. The free and protein-bound SH contents were determined in these tissues. In addition, plasma lactate dehydrogenase levels were determined. Our results showed that methyl parathion exposure significantly lowers the free and protein-bound SH levels in rat tissues. However, lactate dehydrogenase activity in the blood plasma did not display any differences compared to the control group. The free SH concentrations in the control rat liver, brain, and kidney tissues were 3.78 ± 0.1 μmol/100 mg tissue, 1.56 ± 0.08 μmol/100 mg tissue, and 2.16 ± 0.08 μmol/100 mg tissue, respectively, whereas the free SH concentrations in rats exposed to methyl parathion were determined as 0.536 ± 0.1 μmol/100 mg tissue in the liver, 1.06 ± 0.1 μmol/100 mg tissue in the brain, and 0.108 ± 0.03 μmol/100 mg tissue in the kidney. The protein-bound SH concentrations in the liver and in the kidney in rats exposed to methyl parathion displayed a significant decrease also. However, the protein-bound SH level in the brain did not change significantly. These results indicate that methyl parathion exposure partially depletes the free and protein-bound SH levels. Thus, it was concluded that methyl parathion toxicity may partly result from oxidative stress. [ABSTRACT FROM AUTHOR]

Abstract: We investigated effects of prolonged aerobic exercise and severe hyperthyroidism on indices of oxidative damage, susceptibility to oxidants, and respiratory capacity of homogenates from rat liver, heart and skeletal muscle. Both treatments induced increases in hydroperoxide and protein-bound carbonyl levels. Moreover, the highest increases were found when hyperthyroid animals were subjected to exercise. These changes, which were associated to reduced exercise endurance capacity, were in part due to higher susceptibility to oxidants of hyperthyroid tissues. Levels of oxidative damage indices were scarcely related to changes in antioxidant enzyme activities and lipid-soluble antioxidant concentrations. However, the finding that, following exercise the scavenger levels generally decreased in liver homogenates and increased in heart and muscles ones, suggested a net shuttle of antioxidants from liver to other tissues under need. Aerobic capacity, evaluated by cytochrome oxidase activity, was not modified by exercise, which, conversely, affected the rates of oxygen consumption of hyperthyroid preparations. These results seem to confirm the higher susceptibility of hyperthyroid tissues to oxidative challenge, because the mechanisms underlying the opposite changes in respiration rates during State 4 and State 3 likely involve oxidative modifications of components of mitochondrial respiratory chain, different from cytochrome aa3. [Copyright &y& Elsevier]

Effects of nicotine, and nicotine + vitamin E on glucose 6-phosphate dehydrogenase (G-6PD) activity in rat muscle, heart, lungs, testicle, kidney, stomach, brain and liver were investigated in vivo and in vitro on partially purified homogenates. Supplementation period was 3 weeks (n = 8 rats per group): nicotine [0.5 mg/kg/day, intraperitoneal ( ip )]; nicotine + vitamin E [75 mg/kg/day, intragastric ( ig )]; and control group (receiving only vehicle). The results showed that nicotine (0.5 mg/kg, ip ) inhibited G-6PD activity in the lungs, testicle, kidney, stomach and brain by 12.5% (p [ABSTRACT FROM AUTHOR]

Abstract: The expression of two different transcripts for CaV1.2 in rat tissues mirrors that which has previously been described for human tissue, in that expression of transcripts expressing exon 1a is predominant only in heart, whereas expression of transcripts expressing exon 1b is greater in smooth muscle rich tissues such as aorta and intestine. Transcripts expressing exon 1b also predominate in brain and in diaphragm. Western blots indicate that the N-terminus coded for by exon 1b is present in much of the protein in all these tissues except heart. The promoter just upstream of exon 1b has been cloned, sequenced and utilized to drive expression of luciferase in smooth muscle A7r5 cells, cardiac HL-1 cells, skeletal muscle L6 cells and neuronal PC12 cells. The nucleotide sequence of the promoter exhibits 80% identity with the equivalent promoter previously identified in humans and 94% identity with the sequence of the equivalent region of the mouse genome. Evidence in favor of still another promoter upstream of exon 2 has been uncovered. [Copyright &y& Elsevier]

Abstract: The primary objective of this study was to investigate the propensity of Fenvalerate (FEN), a synthetic pyrethroid to induce oxidative stress (OS) in various tissues of growing male rats following a short-term (28 days) dietary regimen and its possible attenuation by dietary (10%) sesame oil (SO). FEN incorporated diet was fed to weanling male rats at the dosages of 0, 250, 500 and 1000ppm. Terminally, significant induction of OS in liver, thymus, spleen and erythrocytes was noticed at higher doses as evidenced by the elevated levels of lipid peroxidation (LPO). Significant dose-dependent depletion of GSH levels, perturbations in antioxidant enzymes, and enhanced protein carbonyls further confirmed the potential of FEN to induce OS in hepatic tissue. In addition, FEN also caused significant increases in activities of hepatic transaminases, ALP and LDH. Interestingly, dietary SO significantly attenuated FEN-induced oxidative damage in liver and other tissues. The degree of protection was remarkably high, since LPO and GSH status, protein carbonyl content and antioxidant defenses in liver and other tissues were brought down to basal levels in the SO+FEN1000 group. These results clearly indicate the potential of FEN to induce oxidative damage in vivo and also suggest the ability of SO, a dietary fat to significantly offset the oxidative damage which may related to the presence of antioxidant compounds in the oil. [Copyright &y& Elsevier]

In this work, we investigated whether cold exposure-induced hyperthyroidism increases oxidative damage and susceptibility to oxidants of rat liver, heart and skeletal muscle. All tissues exhibited gradual increases in hydroperoxide and protein-bound carbonyl levels. Glutathione peroxidase activity increased in all tissues after 2 days and further increased in the muscle after 10 days of cold exposure. Liver glutathione reductase activity increased after 10 days of cold exposure, while heart and muscle activities were not modified. Vitamin E levels were not affected by cold, while coenzyme Q9 and coenzyme Q10 levels decreased in heart and muscle after 2-day cold exposure and were not further modified after 10 days. Liver coenzyme Q9 levels increased after 2 days whereas coenzyme Q10 levels increased after 10 days in the cold. The whole antioxidant capacity was lowered, while parameters positively correlated with susceptibility to oxidants were increased by cold. Lipid fatty acid composition was modified in all tissues. In particular, fatty acid unsaturation degree increased in heart and muscle. Cytochrome oxidase activity increased, suggesting an increased content of hemoproteins, which are able to generate ⋅OH radical. This view was supported by the observation that the tissue susceptibility to H2O2 treatment, which is strongly correlated to iron–ligand content, increased after cold exposure.In this frame, it is apparent that the increase in oxidative capacity, necessary for homeotherm survival in low temperature environments, has potential harmful effects, because it results in increased susceptibility to oxidative challenge. [Copyright &y& Elsevier]

Dipeptidyl peptidase III (DPP III) is a cytosolic zinc-exopeptidase involved in the intracellular protein catabolism of eukaryotes. Although inhibition by thiol reagents is a general feature of DPP III originating from various species, the function of activity important sulfhydryl groups is still inadequately understood. The present study of the reactivity of these groups was undertaken in order to clarify their biological significance.The inactivation kinetics of human and rat DPP III by sulfhydryl reagent p-hydroxy-mercuribenzoate (pHMB) was monitored by determination of the enzyme’s residual activity with fluorimetric detection.Inactivation of this human enzyme exhibited pseudo-first-order kinetics, suggesting that all reactive SH-groups have equivalent reactivity, and the second-order rate constant was calculated to be 3523±567 M−1 min−1. Rat DPP III was hyperreactive to pHMB and showed biphasic kinetics indicating two classes of reactive SH-groups. The second-order rate constants of 3540 M−1 s−1 for slower reacting sulfhydryl, and 21,855 M−1 s−1 for faster reacting sulfhydryl were obtained from slopes of linear plots of pseudo-first-order constants versus reagent concentration. Peptide substrates protected both mammalian DPPs III from inactivation by pHMB. Physiological concentrations of biological thiols and H2O2 inactivated the rat DPP III. Human enzyme was resistant to H2O2 attack and less affected by reduced glutathione (GSH) than the rat homologue. A significantly lower DPP III level, determined by activity measurement and Western blotting, was found in the cytosols of highly oxygenated rat tissues.These results provide kinetic evidence that cysteine residues are involved in substrate binding of mammalian DPPs III. [Copyright &y& Elsevier]

In order to test the hypothesis that the ageing-related alteration in membrane lipids might reflect the biological age of rodents, we studied the effects of age in ad libitum fed (AL) and food-restricted (FR) male Sprague-Dawley rats on the levels of dolichol in different organs involved [liver (L) and kidney (K)] or not involved [brain (B), sciatic nerve (SN), heart (H), soleus (S) and extensor digitorum longus (EDL) muscles] in dolichol excretion. At the given age, tissue dolichol was extracted and assayed by HPLC procedure. Results show that the levels of dolichol were significantly different in different tissues and increased dramatically with increasing age. The anti-ageing FR regimen had significant preventive effects on dolichol accumulation in the excretory organs. The effect of FR on the liver was much bigger than that of kidney. The effect of FR retarding dolichol accumulation in the liver co-varied with the effects of FR on longevity. In conclusion, these data show that the quantity of dolichol in the hepatic tissue might be used as a marker of the biological age of the animal. [ABSTRACT FROM AUTHOR]

Sulphite oxidase (EC 1.8.3.1) is a molybdopterin-containing enzyme that catalyses the oxidation of sulphite to sulphate. Lack of active enzyme produces severe neurodegeneration and early death in humans, showing its essential role. Despite this, the expression of the sulphite oxidase gene in human and rat tissues (especially the brain) has not been elucidated. We therefore examined these tissues and found that the human liver, kidney, skeletal muscle, heart, placenta, and brain showed substantial expression while thymus, spleen, peripheral blood leucocytes, colon, small intestine, and lung showed little expression in humans. In rat, the liver, kidney, heart, brain, and lung (but not skeletal muscle) revealed a hybridization signal with the strongest signal in the liver. The spleen and testis also showed little expression. The differential expression of sulphite oxidase gene in various human brain regions was studied. Expression was seen in all brain regions examined (cerebellum, cerebral cortex, medulla, spinal cord, occipital pole, frontal lobe, amygdala, caudate nucleus, corpus callosum, hippocampus, thalamus, temporal lobe, and putamen). The cerebral cortex showed the highest level of expression. [Copyright &y& Elsevier]

Gene expressions of α-amylase isozymes in rat tissues were analyzed by a reverse transcription-polymerase chain reaction (RT-PCR), followed by EcoRI digestion. This procedure is based on evidence that an RT-PCR product from mouse pancreas RNA is sensitive to EcoRI, but not the product from the salivary gland or liver RNAs. The method was applied to the analysis of α-amylase expression in rat liver after partial hepatectomy, in which a potent expression of pancreas type isozyme was observed. However, no expression of the pancreatic isozyme in the regenerating liver was found. We also analyzed the expression of α-amylase gene in several additional rat tissues. In intestine, stomach, testis and skeletal muscle, the corresponding PCR products were amplified, but few were detected in heart or spleen. Intestine and stomach expressed a pancreatic isozyme of α-amylase. Analyses of the α-amylase activity and protein indicated the presence of the enzyme in those tissues. Immunohistochemical analysis also indicated that the amylase proteins were specifically present in epithelial cells of rat intestinal mucosa. This is a convenient method for identification of α-amylase isozyme mRNA in rodent tissues. [Copyright &y& Elsevier]

Potassium canrenoate (PC), a competitive aldosterone antagonist previously found to increase tumor incidence in rats and to produce genotoxic effects in in vitro systems, was examined in rats to acquire information on its genotoxic activity in vivo. Intragastric administration of 1/2 LD50 produced, as revealed by the Comet assay, a modest but statistically significant increase in the frequency of DNA lesions in liver but not in thyroid and bone marrow of male rats, and in thyroid and bone marrow but not in liver of female rats. In contrast with the frankly positive responses observed in primary cultures of rat hepatocytes (Martelli et al., Mutagenesis 14 (1999) 463–472) any evidence of DNA repair and micronuclei formation was absent in liver of rats treated with 1/2 LD50, and initiation of enzyme-altered liver preneoplastic lesions did not occur in the liver of rats given 100 mg/kg PC once a week for 6 successive weeks. A high and dose-dependent frequency of DNA lesions was found to occur in testes and ovaries of rats given single doses ranging from 1/8 to 1/2 LD50. [Copyright &y& Elsevier]

Knowledge on Bi metabolism in laboratory animals refers to studies at "extreme" exposures, i.e. pharmacologically relevant high-doses (mg kg−1 b.w.) in relation to its medical use, or infinitesimal doses (pg kg−1b.w.) concerning radiobiology protection and radiotherapeutic purposes. There are no specific studies on metabolic patterns of environmental exposure doses (ultratrace level, μg kg−1 b.w.), becoming in this context Bi a "heavy metal fallen into oblivion". We previously reported the results of the metabolic fate of ultratrace levels of Bi in the blood of rats [ 1 ]. In reference to the same study here we report the results of the retention and tissue binding of Bi with intracellular and molecular components. Animals were intraperitoneally injected with 0.8 μg Bi kg−1 b.w. as 205+206Bi(NO) 3 , alone or in combination with 59Fe for the radiolabeling of iron proteins. The use of 205+206Bi radiotracer allowed the determination of Bi down to pg fg−1 in biological fluids, tissues, subcellular fractions, and biochemical components isolated by differential centrifugation, size exclusion chromatography, solvent extraction, precipitation, immunoprecipitation and dialysis. At 24 h post injection the kidney contained by far the highest Bi concentration (10 ng g−1 wt.w.) followed by the thymus, spleen, liver, thyroid, trachea, femur, lung, adrenal gland, stomach, duodenum and pancreas (0.1 to 1.3 ng g−1 wt.w.). Brain and testis showed smaller but consistently significant concentrations of the element (0.03 ng g−1 wt.w). Urine was the predominant route of excretion. Intracellularly, liver, kidney, spleen, testis, and brain cytosols displayed the highest percentages (35%–58%) of Bi of homogenates. Liver and testis nuclei were the organelles with the highest Bi content (24 % and 27 %). However, when the recovered Bi of the liver was recorded as percent of total recovered Bi divided by percent of total recovered protein the lysosomes showed the highest relative specific activity than in other fractions. In the brain subcellular fractions Bi was incorporated by neuro-structures with the protein and not lipidic fraction of the myelin retaining 18 % of Bi of the total homogenate. After the liver intra-subcellular fractionation: (i) 65 % of the nuclear Bi was associated with the protein fraction of the nuclear membranes and 35 % with the bulk chromatin bound to non-histone and DNA fractions; (ii) about 50 % of the mitochondrial Bi was associated with inner and outer membranes being the other half recovered in the intramitochondrial matrix; (iii) in microsomes Bi showed a high affinity (close to 90 %) for the membranous components (rough and smooth membranes); (iv) In the liver cytosol three pools of Bi-binding proteins (molecular size > 300 kDa, 70 kDa and 10 kDa) were observed with ferritin and metallothionein–like protein identified as Bi-binding biomolecules. Three similar protein pools were also observed in the kidney cytosol. However, the amount of Bi, calculated in percent of the total cytosolic Bi, were significantly different compared to the corresponding pools of the liver cytosol. At the best of our knowledge the present paper represents the first in vivo study, on the basis of an environmental toxicology approach, aiming at describing retention and binding of Bi in the rat at tissue, intracellular and molecular levels. [ABSTRACT FROM AUTHOR]