Hepatocyte growth factor (HGF), first identified as a potent mitogen for mature hepatocytes, has been reported to have various activities. We investigated protective effect of continuous HGF supply on carbon tetrachloride (CCl4)-induced acute liver injury in rats. We transfected immortalized but not tumorigenic rat fibroblasts (Rat-1) with an expression plasmid containing the human HGF cDNA and established several cell lines expressing HGF. The biological activity of HGF produced by these cell lines was confirmed by its mitogenic effect on rat hepatocytes in vitro. Either one of the high-HGF-producer cell lines or parental Rat-1 cell line was transplanted into a syngenic rat spleen. Twelve days after transplantation, each rat was intraperitoneally injected with CCl4 and sacrificed 48 h after CCl4 injection. In rats with continuous HGF supply significantly lower serum glutamic-pyruvic transaminase (GPT) level was observed compared to its marked elevation in control rats and the degree of hepatocyte damage was slight on histological analysis. These results indicate that continuous HGF supply effectively inhibits CCl4-induced acute liver injury and may suggest the possibility that this system would be useful on various liver diseases.

It has been hypothesized that blood infusion of reconstituted HDL (rHDL) is a possible therapeutic strategy for the treatment of coronary artery disese. To compare short-term anti-inflammatory activity of wildtype (WT) apoA-I and point mutants, each rHDL containing WT, V156K, or R173C was infused into apo-E deficient atherosclerotic mice. Each rHDL was injected via the tail vein at a dosage of 120 mg/kg of body weight in 0.4 ml of tris-buffered saline (TBS), and blood was then collected at 24 and 48 h post-injection. Although regression activity was observed in each of the rHDL infused groups, a 30% reduction in the lipid-stained area of the aortic sinus was observed in the V156K and R173C-rHDL groups when compared to that of the WT-rHDL group, and this reduction was well correlated with an approximately 60% reduction in the accumulation of macrophages in the lesion area. Additionally, the groups that received the V156K and R173C-rHDL treatments showed smaller increases in the GOT, GPT, interleukin-6, myeloperoxidase (MPO) and lipid hydroperoxide (LPO) serum levels than those that received the WT-rHDL treatment. In addition, the strongest serum paraoxonase and ferric reducing ability was observed in the V156K and R173C-rHDL groups. In vitro nitration and chlorination of apoA-I by MPO treatment revealed that V156K-rHDL and R173C-rHDL were less susceptible to chlorination. Furthermore, rHDL treatment inhibited cellular uptake of oxidized LDL by macrophage cells and the production of proatherogenic species in culture media. In conclusion, blood infusions of the rHDLs exerted in vivo regression activity with anti-inflammatory and antioxidant activity in apo-E deficient mice and THP-1 cells, especially in those that were treated with V156K and R173C apoA-I.

Clotrimazole, a poorly water-soluble antimycotic agent, is a promising therapeutic agent for various diseases including cancer and sickle cell anemia. The oral bioavailability and hepatic toxicity of clotrimazole were compared with its beta-cyclodextrin inclusion form which was prepared by the spray-drying method. The inclusion complex gave significantly higher initial plasma concentrations, Cmax and AUC than did clotrimazole alone, indicating that the drug from the inclusion compound could be more easily absorbed in rats. Furthermore, mice treated with the inclusion compound showed significantly higher GOT/GPT values compared to clotrimazole alone. The inclusion compound also induced hypertrophy of hepatic cells by fat accumulation and disappearance of hepatic sinusoids, indications of pathological changes of liver, suggesting that the inclusion compound could induce more severe tissue damage in the liver than clotrimazole alone. Thus, hepatotoxicity of clotrimazole seems to be correlated with the enhanced oral bioavailability by inclusion complexation. Our results suggest that, in the development of a novel oral product, appearance or enhancement of hepatic toxicity must be considered along with oral bioavailability.

Imbalances in the intracellular nucleotide precursor pools in mammalian cells can result in the induction of mutations during the DNA replication process. By using a shuttle vector system developed in our laboratory, we have analyzed the sequence specificity of mutations induced in mouse A9 cells by exposure of the cells to a high concentration of thymidine. The target for mutagenesis in these studies was the bacterial gpt gene stably integrated into the chromosomal DNA of the mouse cells. Previous studies in this laboratory had generated a large panel of xanthine guanine phosphoribosyl-transferase (EC 2.4.2.22)-negative mutant lines that possess single-base mutations within the gpt coding sequence. This study utilized four xanthine guanine phosphoribosyltransferase-negative mutant lines to assess the frequency of mutation induced by thymidine at guanine residues in four sequence contexts: the 5' and 3' guanine residues of a GG doublet, the middle guanine residue of a GGG triplet, and the 3' guanine residue of a GGGG quartet. The results of this study demonstrate that treatment of cultured cells with a high concentration of thymidine can result in G.C----A.T transition mutations that occur preferentially at the 3' guanine residue of a run of two or more adjacent guanines. Guanine residues flanked on their 3' side by other guanine residues are severalfold less mutable by thymidine than are guanine residues flanked on their 3' side by a different base. This study demonstrates a sequence-specific mode for thymidine-induced mutations and suggests implications for mutagenesis in vivo.

The efficiency of a monomolecular film of (3-glycidoxypropyl) trimethoxysilane (GPTS) on a shear horizontal guided (Love) acoustic wave immunosensor to detect whole Escherichia coli (E. coli) bacteria is demonstrated. Direct anti-E. coli antibodies grafting onto the sensor surface did not lead to a significant bacteria immobilisation, partially attributed to the SiO2 sensor surface roughness. An innovative method has been set up to get around this difficulty and to detect whole bacteria. It consists in grafting goat anti-mouse antibodies (GAM) onto the sensor surface in a first step and introducing E. coli bacteria mixed with anti-E. coli antibodies onto the sensor in a second step. We describe the characteristics of such a technique like sample preparation time (lower than 30 min) and temperature improvements. A 37 degrees C experimental temperature led to the fastest bacteria binding kinetic, reducing the total analysis time. This method enables to keep the specificity of the antibody/antigen interaction and provides significant results in less than 1h. This leads to a detection threshold of 10(6) bacteria/ml in a 500 microl chamber.

We wished to determine whether simian virus 40 (SV40)-transformed xeroderma pigmentosum cells, despite their defective DNA repair, were suitable for DNA-mediated gene transfer experiments with linked genes. Expression of a nonselectable gene (cat, coding for chloramphenicol acetyltransferase [CAT]) linked to a selectable gene (gpt, coding for xanthine-guanine phosphoribosyltransferase [XPRT]) in the plasmid pSV2catSVgpt was quantified after transfection of SV40-transformed xeroderma pigmentosum [XP20s(SV40)] and normal human [GM0637(SV40)] fibroblast cell lines. A novel autoradiographic assay with [3H]xanthine incorporation showed 0.5 to 0.7% phenotypic expression of XPRT in both cell lines. Without selection, transient CAT activity was 20 times greater in the GM0637(SV40) than in the XP20s(SV40) cells, and transient XPRT activity was 5 times greater. Both of these transient activities were increased and equalized in both cell lines by transfection with pRSVcat or pRSVgpt. Genotypic transformation to gpt+ occurred at a frequency of 2 X 10(-4) to 4 X 10(-4) in both cell lines with pSV2catSVgpt. After 2 to 3 months in selective medium, stable expression of the (nonselected) cat gene was found in 11 (92%) of 12 gpt-containing clones derived from GM0637(SV40) cells and in 13 (81%) of 16 gpt-containing clones from XP20s(SV40) cells. However, the levels of CAT activity did not correlate with those of XPRT activity, and both of these activities varied more than 100-fold among different clones. Copies (1 to 4) of the gpt gene were integrated in four clones of the GM0637(SV40) cells having an XPRT activity of 1 to 5 nmol/min per mg, but 5 to 80 copies were integrated in four XP20s(SV40) clones with an XPRT activity of 0.8 to 1.8 nmol/min per mg. This study shows that XP20s(SV40) is as suitable for gene transfer experiments as the normal human line GM0637(SV40).

All retroviral genomes contain a nucleotide sequence designated as the primer binding site (PBS) which is complementary to the tRNA used for initiation of reverse transcription. For human immunodeficiency virus type 1 (HIV-1), all naturally occurring genomes have a PBS complementary to tRNA3Lys. However, within HIV-1 virions, there are approximately equal amounts of tRNA1Lys, tRNA2Lys, and tRNA3Lys. We have used an endogenous reverse transcription-PCR technique specific for the tRNA species within isolated HIV-1 virions to demonstrate that in addition to tRNA3Lys, tRNA1Lys and tRNA2Lys could be used for initiation of HIV-1 reverse transcription. Using a single-round infection assay which employed an HIV-1 genome with a gpt gene encoding xanthine-guanine phosphoribosyl transferase in place of the env gene, we generated cell lines resistant to mycophenolic acid. Analysis of the U5-PBS from single-cell clones revealed PBS complementary to tRNA3Lys, not tRNA1Lys or tRNA2Lys. A mutant HIV-1 genome was then created which would favor the completion of reverse transcription with tRNA1,2Lys. Using this provirus in the complementation system, we again found only genomes with a PBS complementary to tRNA3Lys from proviral DNA isolated from gpt-resistant single-cell colonies. Finally, infection of cells with a mutant HIV genome with a PBS complementary to tRNA1,2Lys resulted in gpt- resistant cell colonies which contained integrated provirions with a PBS complementary to tRNA1,2Lys. The results of these studies suggest that the selection of tRNA3Lys for initiation of HIV-1 reverse transcription occurs both at the initiation and at a postinitiation step in reverse transcription prior to integration of the proviral DNA.

BACKGROUND

Genotoxicity is often a prerequisite to the development of malignancy. Considerable evidence has shown that exposure to asbestos fibers results in the generation of chromosomal aberrations and multilocus mutations using various in vitro approaches. However, there is less evidence to demonstrate the contribution of deletions to the mutagenicity of asbestos fibers in vivo.

OBJECTIVE

In the present study, we investigated the mutant fractions and the patterns induced by chrysotile fibers in gpt delta transgenic mouse primary embryo fibroblasts (MEFs) and compared the results obtained with hydrogen peroxide (H2O2) in an attempt to illustrate the role of oxyradicals in fiber mutagenesis.

RESULTS

Chrysotile fibers induced a dose-dependent increase in mutation yield at the redBA/gam loci in transgenic MEF cells. The number of lambda mutants losing both redBA and gam loci induced by chrysotiles at a dose of 1 microg/cm(2) increased by>> 5-fold relative to nontreated controls (p < 0.005). Mutation spectra analyses showed that the ratio of lambda mutants losing the redBA/gam region induced by chrysotiles was similar to those induced by equitoxic doses of H2O2. Moreover, treatment with catalase abrogated the accumulation of y-H2AX, a biomarker of DNA double-strand breaks, induced by chrysotile fibers.

CONCLUSIONS

Our results provide novel information on the frequencies and types of mutations induced by asbestos fibers in the gpt delta transgenic mouse mutagenic assay, which shows great promise for evaluating fiber/particle mutagenicity in vivo.

Inhibition of poly(ADP-ribosylation) reduces random genomic integration of transfected DNA and mildly stimulates intrachromosomal homologous recombination in mammalian cells. We investigated the effect of inhibition of poly(ADP-ribosylation) on the efficiency of gene targeting in Chinese hamster ovary (CHO) cell line ATS-49tg. This cell line is hemizygous for a defective adenine phosphoribosyltransferase (aprt) gene and is hypoxanthine phosphoribosyltransferase (hprt) deficient. Plasmid pAG100 contains a portion of the CHO aprt gene sufficient to correct the defect in ATS-49tg cells via gene targeting; pAG100 also contains an Escherichia coli guanine phosphoribosyltransferase (gpt) gene. Following transfection of ATS-49tg cells with pAG100, selection for gpt-positive transfectants allowed recovery of cells that had randomly integrated pAG100 while selection for aprt-positive cells allowed recovery of cells that had undergone gene targeting at the endogenous aprt locus. Treatment of cells with 3 mM 3-methoxybenzamide (3-MB), an inhibitor of poly(ADP-ribose) polymerase, decreased random integration and gene targeting of electroporated pAG100 about 5-fold. In contrast, treatment with 3 mM 3-MB during calcium phosphate transfection could reduce random integration more than 150-fold while reducing gene targeting less than two-fold. Therefore, as much as a 100-fold enrichment for gene targeting was achieved with calcium phosphate transfection.

Chronic administration of beta-sitosterol subcutaneously to rats for 60 days was well tolerated and there was no clear cut evidence of any gross or microscopic lesions either in the liver or kidney. Liver and kidney function tests were assessed by determining the blood/serum parameters like haemoglobin, blood glucose, serum protein, serum bilirubin, serum cholesterol, serum GPT and serum GOT. All the parameters were in the normal range except serum protein and serum cholesterol. Serum cholesterol was the only variable which depleted markedly in both sexes in a dose-dependent manner suggesting intrinsic hypocholesterolemic effect of the sterol.

The present study was undertaken to investigate toxic potentials of linoleic acid anilide (LAA) and heated linoleic acid anilide (HLAA) and their possible role in the etiology of toxic oil syndrome (TOS). Male Sprague-Dawley rats were given 250 mg/kg of LAA or HLAA in mineral oil through gavage, on alternate days for 2 weeks (total 7 doses). Control rats received an equal volume of vehicle only. The animals were sacrificed at day 1, 7 and 28 following the last dose. Ratio of organ weight/body weight showed a significant increase in lung in LAA group at day 7 while spleen showed remarkable increases in both treatment groups at day 1 and 7. On the other hand, this ratio showed decreases in case of liver, brain and heart at some time points. Among blood parameters, red cell counts and hemoglobin content decreased at day 1 in both LAA and HLAA treated groups, while platelet counts showed an increase. Serum LDH, GOT and GPT activities significantly decreased at day 1 and 7 in both LAA and HLAA treated groups, however, these changes were more prominent in the HLAA treated group. Interestingly, at day 28, these serum enzyme levels recovered to control levels. Both LAA and HLAA treated groups showed a decrease in serum IgM levels at day 1, however, at day 7 only the LAA group showed a significant decrease. IgA levels significantly increased in both groups at all the time points studied and were more pronounced in the HLAA treated group. Similarly, IgG levels also showed increases in both the groups. In addition to serum immunoglobulin changes, alterations in the lymphocyte subpopulations were also observed. While T-cell population decreased, B-cell population remained unchanged. Among T-cell subsets, T-helper cells did not show any change while T-suppressor cells decreased significantly at day 1 in the LAA group and at day 1 and 7 in the HLAA group, but regained control levels at day 28. The biochemical and immunological alterations observed in this study as a result of LAA and HLAA exposure and more so by HLAA further support that the fatty acid anilides may play a role in the etiology of TOS.

We utilized a plasmid vector host cell reactivation assay to probe the biological functioning of DNA expression vectors and their encoded genes. We studied the effect of ultraviolet radiation or acid-heat treatment on the transient expression of genes transfected into normal human cells and into DNA repair deficient (xeroderma pigmentosum) cells and modification of gene expression by sodium butyrate. U.v. inactivation of transient expression of the bacterial gpt gene contained in a non-replicating expression vector plasmid, pSV2catSVgpt, was much greater in three xeroderma pigmentosum lines than in the four other human cell lines tested. In contrast, treatment of pSV2catSVgpt with acid and heat to produce apurinic sites resulted in a similar slope of the inactivation curve of the bacterial cat gene in the repair deficient and repair proficient cells. Thus, u.v. damage of DNA expression vectors was subject to repair by the normal host cells, but acid-heat treatment resulted in damage (apurinic sites) that was handled in a similar manner by excision repair deficient and excision repair proficient human cells. In both normal and xeroderma pigmentosum cells sodium butyrate treatment of cells resulted in a greater stimulation of chloramphenicol acetyltransferase expression with u.v. damaged than with undamaged plasmid. This assay thus permits examination of the effects of defined types of DNA damage on plasmid expression and study of its modulation by cellular repair activities.

A phase I study of recombinant human tumor necrosis factor (rHu-TNF:PT-050) given by intravenous infusion over 30 min or by intratumoral administration was performed in 53 patients with various types of malignant tumors. The dose of rHu-TNF was started with 0.1 x 10(6) U/body for both intravenous infusion and intratumoral administration and increased to 5 x 10(6) U/body for intravenous infusion and 2 x 10(6) U/body for intratumoral administration. The side effects of rHu-TNF given by intravenous infusion included fever, shaking chills, hypotension, general malaise, nausea, and vomiting, and clinical laboratory tests showed elevations of GOT, GPT, and ALP, etc. Among these, only hypotension was dose-related and was considered to be a dose-limiting factor. The maximum tolerable dose estimated was 1 x 10(6) U/body. The plasma concentration of rHu-TNF after completion of a 30-min infusion was dose-dependent, and the elimination half-life was 0.5-2.4 hr. When the rHu-TNF was administered intratumorally, the frequency of side effects was low compared with intravenous infusion.

Seabream and turbot juveniles (40-520 g) were exposed to constant exogenous NH5-N concentrations (1.27-4.27 mmol/l; pH, 8.15). In 96 hr acclimated fish, plasma TA-N (total ammonia nitrogen) contents were positively correlated to ambient ammonia concentrations. The LD50 were 2.2-2.5 mmol/l TA-N in both species. There were no marked osmoregulatory disturbances and plasma urea-N, thyroid hormones levels and gill (Na-K)-ATPase activities were only affected at the highest concentrations. Liver GOT, GPT and GIDH activity dose-response were low and species dependent. In cannulated and non-cannulated turbot exposed to half 96 hr LC50 (lethal ambient concentration for 50% of the population), there was a rapid, pronounced and prolonged increase in plasma TA-N, followed by an immediate decline when exogenous ammonia supply was stopped. Maximum loading and unloading were observed within 1-3 hr. Plasma cortisol levels indicated a stressful situation in exposed fish (150 ng/ml) and a quick recovery capacity. In dose and time response experiments, the most relevant physiological indicator of ammonia stress was blood TA-N content. Other parameters tested led either to transient or low amplitude responses except when fish approached death.

In order to identify possible signatures of the most typical structures adopted by guanine-rich oligonucleotides, we submitted them to the crossed fire of circular dichroism (CD) and electrophoresis. These signatures show up in the circular dichroism spectra even when simultaneously present within the same molecule. Guanine-rich oligonucleotides, when structured, manifest themselves by CD contributions around 260 or 295 nm. For instance, positive bands at 264 nm and 295 nm, respectively, signal the parallel and antiparallel guanine quartets, while a positive band around 261 nm may indicate the presence of a (parallel?) Hoogsteen duplex. A positive band at 264 nm may also reflect the presence of rigidly and unusually oriented GpT and TpG steps within loops. The signatures are additive with those of other structural features of the same molecule, such as hairpins or Watson-Crick duplexes, whose bands are observed at 280 nm.

pSV2gpt-Transformed and wild-type Chinese hamster ovary (CHO) cell lines have been used to study radiation-induced mutation at the molecular level. The transformant, designated AS52, was constructed from a hypoxanthine-guanine phosphoribosyl transferase (HPRT)-deficient CHO cell line and contains a single, functional copy of the Escherichia coli xanthine-guanine phosphoribosyl transferase (XPRT) gene (gpt) stably integrated into the Chinese hamster genome. AS52 and wild-type CHO-K1-BH4 cells exhibit similar cytotoxic responses to uv light and X rays; however, significant differences occur in mutation induction at the gpt and hprt loci. A number of HPRT and XPRT mutants which arose following irradiation were analyzed by Southern-blot hybridization. Most XPRT (21/26) and all HPRT (23/23) mutants induced by uv light exhibited hybridization patterns indistinguishable from their parental cell lines. In contrast, all XPRT (26/26) and most HPRT mutants (15/21) induced by X irradiation contained deletion mutations affecting some or all of the gpt and hprt loci, respectively. These results indicate that X rays induce predominantly deletion mutations, while uv light is likely to induce point mutations at both loci.

A 51-year-old woman was evaluated because of Raynaud's phenomenon, Sjögren's syndrome, and general malaise. There was neither skin itching nor jaundice. Endoscopic retrograde cholangiopancreatography showed a normal extrahepatic as well as intrahepatic biliary tree. Serum GOT and GPT fluctuated with episodes of marked increases. The alkaline phosphatase was slightly increased and total cholesterol was normal. There were marked increases of IgG and IgM. AMA was positive at a titer of 1:320, which was measured by an indirect immunofluorescence method. PBC-specific AMA (anti-M2) was positive, but mixed-form AMA (anti-M4) negative. An LE-cell test, ASMA, ANA, and anti-DNA antibody were all positive on several repeated tests. A wedge liver biopsy tissue showed mixed features of PBC and CAH. A diagnosis of CAH was made on the basis of the clinical, serological, and morphologic findings. The patient responded well to prednisolone treatment with prominent improvement of her symptoms and liver function tests. Subsequently, AMA fell to undetectable levels by indirect immunofluorescence method.

To define a selective system for the study of rat tyrosine aminotransferase (TAT; EC 2.6.1.5) gene expression, we have introduced into cultured cells the selectable bacterial gene gpt linked to TAT gene flanking sequences. After integration in host cell DNA, the chimeric gene exhibits the same pattern of regulation as the TAT gene. In hepatoma cells, its expression is induced after glucocorticoid hormone treatment and repressed after fusion with fibroblasts. In fibroblasts, the chimeric gene is not expressed. The correct pattern of regulation is lost when the number of integrated copies is high. At copy number above 10, the transfected gene responds poorly to glucocorticoids in hepatoma cells. At copy number above 50, the gene is expressed in fibroblasts. Another gene present in the same construction and controlled by the SV40 early promoter and enhancer is positively regulated by glucocorticoids in hepatoma cells but not after fusion with fibroblasts. These data indicate that in hybrid cells, both TAT promoter and glucocorticoid-responsive elements are negatively regulated.

Sixty male workers in a lindane (gamma-hexachlorocyclohexane)-producing factory were examined with regard to health in comparison with an external control group of 20 clerks. Case history, physical examination, neurologic status, and ECG revealed no significant differences between groups. However, the following significant differences in clinical-chemical blood tests were ascertained: higher polymorphonuclear leukocyte count, lower lymphocyte count, higher reticulocyte count, lower prothrombin (Quick's) test, and lower blood concentrations of creatinine and uric acid. No significant differences were observed in total red and white blood cell as well as platelet counts, hemoglobin content, the other counts of differential blood picture, gamma-GT, GOT, GPT, LDH, cholinesterase, triglycerides, cholesterol, and urea. In spite of a pronounced exposure to the alpha-, beta-, and gamma-isomers of hexachlorocyclohexane, no signs of severe impairment of health were observed; only small deviations in some laboratory tests were found having no pathologic significance. However, biological monitoring and health supervision of HCH-exposed workers should be carried out.

The dose- and time-related hepatotoxic effects of acetaminophen were investigated in rats using biochemical parameters as indices of hepatotoxicity supplemented by the histopathological examination of the livers. The acute or subacute (twice daily for 7 days) administration of 0.25 g/kg acetaminophen did not produce any noticeable hepatocellular damage. On the other hand, dose-dependent elevations in serum enzyme glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT) and sorbitol dehydrogenase (SDH) activities and hepatic triglyceride (TG) levels were observed following the administration of single doses of 0.5 and lg/kg acetaminophen. Maximal hepatic damage occurred 12-18 h after acute dosing, while the hepatic function returned to control levels by 48-72 h. In contrast with the acutely treated rats, the serum enzyme activities and the hepatic TG levels remained unchanged following 7-day treatment with 0.5 or 1 g/kg acetaminophen. Also, histopathologically the degree of acetaminophen-induced hepatic necrosis was found to be far less extensive in rats given 0.5 and 1 g/kg acetaminophen twice daily for up to one week, as compared with the animals sacrificed 18 h after administering single equivalent doses of this drug. The results suggest that the liver function is reversibly impaired following acetaminophen overdosage, and that the intensity of acetaminophen-induced hepatotoxicity becomes less severe after repeated exposure to this hepatotoxin.

Plasma components of 6 to 12-month-old beagles were examined using a Technicon auto-analyzer. Age-related changes were noted for 8 of the 21 components: the levels of total protein (T. Pro) and iron (Fe) gradually increased while those of alkaline phosphatase (ALP), creatine phosphokinase (CPK) and inorganic phosphorus (Pi) persistently decreased in both sexes. Triglyceride (Trigly) in female dogs, glutamic-pyruvic transaminase (GPT) and urea nitrogen (Urea-N) in male dogs tended to increase. The following thirteen components showed no significant variation during the period of observation: glucose (Glu), lactic dehydrogenase (LDH), albumin (Alb), creatinine (Crea), glutamic-oxaloacetic transaminase (GOT), leucine aminopeptidase (LAP), total bilirubin (T. Bil), amylase (Amy), total cholesterol (T. Chol), sodium (Na), potassium (K), chloride (Cl) and calcium (Ca). Our results generally agree with the reported findings on beagles from various institutions.