Hormonal regulation of type I insulin-like growth factor receptors of Leydig cells in hypophysectomized rats.

The effects of hCG and various pituitary hormones on type I insulin-like growth factor (IGF) receptors of purified Leydig cells of hypophysectomized rats were studied. The number of type I IGF receptors of Leydig cells obtained from hypophysectomized rats (18.0 +/- 1.5 fmol/10(6) cells) was lower than that in normal rats (54.6 +/- 5.3 fmol/10(6) cells; P less than 0.05). After a single administration of hCG (10 U, ip), specific binding of [125I]IGF-I to purified Leydig cells increased 3-fold. Scatchard analyses of the binding data suggested that increased binding was the result of an increase in receptor number, whereas binding affinity remained unaltered. Type I IGF receptor increased within 12 h and remained persistently elevated 96 h after hCG treatment. Administration of hCG (10 U, ip) daily for 5 days increased type I IGF receptor levels to 73.2 +/- 8 fmol/10(6) cells (P less than 0.001). FSH caused a small but significant increase in type I IGF receptors. Concomitant administration of FSH and hCG further enhanced IGF-I-binding capacity. IGF-I-binding affinity of Leydig cells treated with FSH or FSH plus LH was not significantly different from that in the control hypophysectomized rats. Daily administration of GH for 5 days also upregulated type I IGF receptors, whereas PRL had no effect. FSH, GH, and PRL administration had no effect on serum testosterone levels. Serum testosterone levels increased to 3.99 +/- 0.35 ng/ml after 5 days of treatment with hCG. Concomitant administration of FSH and hCG caused a further increased in serum testosterone levels (6.13 +/- 0.46 ng/ml; P less than 0.01). The present study suggests that type I IGF receptors of Leydig cells can be up-regulated by LH, FSH, and GH. However, hCG/LH seems to be the most important factor in maintaining and regulating type I IGF receptors of Leydig cells. Steroidogenic and growth-promoting effects of hCG and pituitary hormones on Leydig cells may be mediated by increased type I IGF receptors.

Identification of a null allele of CYP2C9 in an African-American exhibiting toxicity to phenytoin.

Cytochrome P450 (CYP) 2C9 is the principal enzyme responsible for the metabolism of numerous clinically important drugs. Two polymorphic alleles CYP2C9*2 and CYP2C9*3 have been documented which affect the metabolism and clinical toxicity of drugs such as phenytoin, warfarin, glipizide, and tolbutamide. The present study reports the first example of a null polymorphism in CYP2C9. This mutation dramatically affects the half-life and clinical toxicity of phenytoin. The study subject was a female African-American presented to the emergency department with phenytoin toxicity evidenced by mental confusion, slurred speech, memory loss and the inability to stand. She exhibited extremely poor clearance of phenytoin with an elimination half-life of approximately 13 days. Genotyping studies demonstrated that the patient did not possess any known variant CYP2C9 alleles. Phenytoin is metabolized to a minor extent by the polymorphic CYP2C19, but this individual did not possess any variant CYP2C19 alleles. Sequencing studies revealed that the individual was homozygous for a new CYP2C9 allele (CYP2C9*6) with the deletion of an adenine at base pair 818 of the cDNA. The clearance of phenytoin in this individual is estimated to be approximately 17% of that observed in normal patients. The frequency of this allele was 0.6% (95% confidence limits of 0.1 to 3.5%) in 79 African-Americans and 0% (95% confidence limits of 0 to 1.1%) in 172 Caucasians. The study also demonstrates the severe clinical consequences to patients with a null mutation in CYP2C9 after treatment with normal doses of phenytoin.

Polymorphisms in human CYP2C8 decrease metabolism of the anticancer drug paclitaxel and arachidonic acid.

Cytochrome P450 (CYP) 2C8 is the principal enzyme responsible for the metabolism of the anti-cancer drug paclitaxel (Taxol). It is also the predominant P450 responsible for the metabolism of arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs) in human liver and kidney. In this study, we describe two new CYP2C8 alleles containing coding changes: CYP2C8*2 has an Ile269Phe substitution in exon 5 and CYP2C8*3 includes both Arg139Lys and Lys399Arg amino acid substitutions in exons 3 and 8. CYP2C8*2 was found only in African-Americans, while CYP2C8*3 occurred primarily in Caucasians. Neither occurred in Asians. The frequency of the CYP2C8*2 allele was 0.18 in African-Americans, and that of CYP2C8*3 was 0.13 in Caucasians. CYP2C8*1 (wild-type), CYP2C8*2 and CYP2C8*3 cDNAs were expressed in Escherichia coli, and the ability of these enzymes to metabolize both paclitaxel and arachidonic acid was assessed. Recombinant CYP2C8*3 was defective in the metabolism of both substrates. The turnover number of CYP2C8*3 for paclitaxel was 15% of CYP2C8*1. CYP2C8*2 had a two-fold higher Km and two-fold lower intrinsic clearance for paclitaxel than CYP2C8*1. CYP2C8*3 was also markedly defective in the metabolism of arachidonic acid to 11,12- and 14,15-EET (turnover numbers 35-40% that of CYP2C8*1). Thus, CYP2C8*3 is defective in the metabolism of two important CYP2C8 substrates: the anticancer drug paclitaxel and the physiologically important compound arachidonic acid. This polymorphism has important clinical and physiological implications in individuals homozygous for this allele.

Pharmacokinetics of chlorpheniramine, phenytoin, glipizide and nifedipine in an individual homozygous for the CYP2C9*3 allele.

Genetic polymorphisms in the cytochrome P450 (CYP) family are widely known to contribute to interindividual differences in the pharmacokinetics of many drugs. Several alleles for the CYP2C9 gene have been reported. Individuals homozygous for the Leu359 variant (CYP2C9*3) have been shown to have significantly lower drug clearances compared with Ile359 (CYP2C9*1) homozygous individuals. A male Caucasian who participated in six bioavailability studies in our laboratory over a period of several years showed extremely low clearance of two drugs: phenytoin and glipizide (both substrates of CYP2C9), but not for nifedipine (a CYP3A4 substrate) and chlorpheniramine (a CYP2D6 substrate). His oral clearance of phenytoin was 21% of the mean of the other 11 individuals participating in the study, and his oral clearance of glipizide, a second generation sulfonylurea structurally similar to tolbutamide, was only 188% of the mean of the other 10 individuals. However, his oral clearance of nifedipine and chlorpheniramine did not differ from individuals in other studies performed at our laboratories. An additional blood sample was obtained from this individual to determine if he possessed any of the known CYP2C9 or CYP2C19 allelic variants that would account for his poor clearance of the CYP2C9 substrates (phenytoin and glipizide) compared with the CYP3A4 (nifedipine) and CYP2D6 (chlorpheniramine) substrates. The results of the genotype testing showed that this individual was homozygous for the CYP2C9*3 allele and did not possess any of the known defective CYP2C19 alleles. This study establishes that the Leu359 mutation is responsible for the phenytoin and glipizide/tolbutamide poor metabolizer phenotype.

An additional defective allele, CYP2C19*5, contributes to the S-mephenytoin poor metabolizer phenotype in Caucasians.

The metabolism of the anticonvulsant drug mephenytoin exhibits a genetic polymorphism in humans. This polymorphism exhibits marked racial heterogeneity, with the poor metabolizer PM phenotype representing 13-23% of oriental populations, but only 2-5% of Caucasian populations. Two defective CYP2C19 alleles (CYP2C19*2 and CYP2C19*3) have been described, which account for more than 99% of Oriental poor metabolizer alleles but only approximately 87% of Caucasian poor metabolizer alleles. Therefore, additional defects presumably contribute to the poor metabolizer in Caucasians. Recent studies have found a third mutation CYP2C19*4, which accounts for approximately 3% of Caucasian poor metabolizer alleles. A fourth rare mutation (CYP2C19*5A) (C99,A991,Ile331;C1297T,Arg433-->Trp) resulting in an Arg433 to Trp substitution in the heme-binding region has been reported in a single Chinese poor metaboliser outlier belonging to the Bai ethnic group. The present study identifies a second variant allele CYP2C19*5B (C99-->T; A991-->G, Ile331-->Val; C1297-T, Arg433-->Trp in one of 37 Caucasian poor metabolizers. The frequency of the CYP2C19*5 alleles is low in Chinese (approximately 0.25% in the Bai ethnic group) and Caucasians (< 0.9%). However, these alleles contribute to the poor metabolizer phenotype in both ethnic groups and increases the sensitivity of the genetic tests for identifying defective alleles to approximately 100% in Chinese poor metabolizers and 92% in Caucasian poor metabolizers genotyped in our laboratory. The Arg433 to Trp mutation in the heme-binding region essentially abolishes activity of recombinant CYP2C19*5A toward S-mephenytoin and tolbutamide, which is consistent with the conclusion that CYP2C19*5 represents poor metabolizer alleles.

Expression of the oxidative base excision repair enzymes is not induced in TK6 human lymphoblastoid cells after low doses of ionizing radiation.

Most of the DNA damage produced by ionizing radiation is repaired by the base excision repair (BER) pathway. To determine whether the BER genes were up-regulated by low doses of ionizing radiation, we investigated their expression in TK6 human lymphoblastoid cells by measuring mRNA levels using real-time quantitative PCR. No induction at the transcriptional level of any of the base excision repair genes, NTH1 (NTHL1), OGG1, NEIL1, NEIL2, NEIL3, APE1, POLB, or accessory protein genes, LIG3, XRCC1 or XPG, was found at gamma-radiation doses ranging from 1 cGy to 2 Gy in a 24-h period. As has been measured in other cell lines, a dose-dependent induction of CDKN1A (WAF1) mRNA levels was observed in TK6 cells in the dose range of 0.5 to 2.0 Gy. We also examined BER enzyme activity on 8-oxoguanine-, dihydrouracil- and furan-containing oligonucleotide substrates and found no increase in extracts of TK6 cells after gamma-ray doses of 0.5-2.0 Gy. These data were corroborated by Western blot analysis of APE1 and NTH1, suggesting that the BER enzymes are also not up-regulated at the post-transcriptional level after ionizing radiation exposure.

Weight loss in frankfurters during thermal processing.

A simplified two-term model was developed to predict the weight loss of frankfurters during thermal processing at various process conditions and with various product compositions. The model was validated with the experimental data. The moisture loss rate was found to be proportional to product temperature and inversely proportional to the fat-protein ratio of the product.

Effects of Tamm-Horsfall glycoprotein and albumin on struvite crystal growth in urine of cats.

Tamm-Horsfall protein (THP) was isolated and purified, using pooled urine collections from clinically normal cats. The effects of feline THP and purified feline serum albumin on urine chemical and struvite crystal variables were compared, using an in vitro crystal growth system and 24-hour samples of pooled urine obtained from 4 cats. Urine samples were placed in wells of cell culture plates, increasing concentrations of ammonium hydroxide were added to adjacent wells to stimulate struvite crystal growth, and the plates were incubated at 37 C. The effect of albumin and THP on crystal growth in sample wells was compared with that in control wells, without protein addition, in the same plate. Crystal growth was assessed by determination of number of crystals and supersaturation index, a scale of crystal habit at different degrees of supersaturation, by use of direct visualization with an inverted microscope. Albumin addition did not have significant effect on either crystal number or supersaturation, compared with controls. Addition of THP significantly (P < 0.05) increased crystal number and supersaturation index. It was concluded that THP significantly (P < 0.05) promoted growth of struvite crystals in feline urine, and thus, may have a role in feline struvite uroliths and struvite urethral plug formation.

Effects of choreito consumption on struvite crystal growth in urine of cats.

The effect of a dietary supplement, choreito, on in vitro struvite crystal growth in feline urine was evaluated. Adult specific-pathogen-free cats (4 females, 4 males) considered to be clinically normal on the basis of physical examination findings and normal results of CBC, serum biochemical analyses, and urinalyses obtained before the beginning of the study were used. Before 24-hour urine sample collections were made, cats were fed a commercial canned diet with 0 or 500 mg of choreito supplement/kg of body weight for at least 2 weeks in a cross-over design with 4 cats/treatment. Filtered urine samples were analyzed for urine pH, specific gravity, osmolality, and urine electrolytes. The struvite activity product was calculated, using a statistical software program that calculates urine saturation. Urine samples were placed in wells of cell culture plates, increasing concentrations of ammonium hydroxide were added to adjacent wells to stimulate struvite crystal growth, and the plates were incubated at 37 C. Crystal growth was assessed by determination of number of crystals and supersaturation index by direct visualization, using an inverted microscope. Supplementation of the diet with choreito (at this concentration) did not change urine pH, specific gravity, osmolality, urine electrolyte composition, or calculated struvite activity product. However, supplementation significantly (P < 0.05) reduced crystal number and supersaturation index. These results indicate that direct observation of struvite crystal formation in whole urine may more accurately predict the effects of treatments to prevent or treat struvite urolithiasis than do calculations based on electrolyte concentration that do not account for the effect of urine macromolecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Moisture mobility in meat emulsion during thermal processing: Analysis of slab moisture profile.

A determination of the moisture distribution in meat emulsion slab at several process times as a function of its composition and temperature, and smokehouse relative humidity, with a technique and model for determining the corresponding moisture mobility are presented. The moisture diffusivity was increasing with the decrease in the fat-protein ratio and increase in the product temperature and moisture concentration and follows an Arrhenius type relationship. The moisture profiles in the meat emulsion during processing were very steep at the product surface.

Effects of choreito and takushya consumption on in vitro and in vivo struvite solubility in cat urine.

OBJECTIVE: To determine the effects of the takushya portion of choreito, a traditional Chinese treatment for urolithiasis, on urine and struvite crystal variables in cats fed diets containing takushya. SAMPLE POPULATION: 6 male and 6 female adult cats, all considered to be clinically normal on the basis of physical examination findings, results of CBC, serum biochemical analyses, urinalyses, and urine cultures; and freedom from urolithiasis on the basis of urethrocystoscopic (females) or urethrocystographic (males) findings. PROCEDURE: Cats were fed a commercial canned diet supplemented with 0.1-mg of takushya/kg of body weight, or with 0.5 mg of choreito/kg. Diets were fed, using a Latin-square design, to 3 groups of 4 cats (2 male, 2 female) each for 2 weeks, followed by blood and 24-hour urine sample collections. RESULTS: Consumption of takushya, which comprises 20% by weight of choreito, was not associated with adverse effects in cats at the amounts provided during the period of study. Moreover, takushya was responsible for most of the effect of choreito consumption on reduction of urine pH, and approximately half its ability to reduce struvite crystal formation in cat urine. CLINICAL RELEVANCE: Alternative treatments for struvite urolithiasis in cats may be feasible.

Effects of choreito consumption on urine variables of healthy cats fed a magnesium-supplemented commercial diet.

OBJECTIVE: To investigate the effect of choreito consumption (500 mg/kg of body weight/d) on struvite crystal formation and signs of lower urinary tract disease (LUTD) in cats consuming a commercial canned diet with 0.5% added inorganic magnesium. SAMPLE POPULATION: 6 male and 6 female adult cats, all considered to be clinically normal on the basis of physical examination findings; results of CBC, serum biochemical analyses, urinalyses, and urine cultures; and freedom from urolithiasis on the basis of urethrocystoscopic (females) or urethrocystographic (males) findings. PROCEDURE: Diets were fed for 12 weeks, or until appearance of signs of LUTD, including dysuria, hematuria, urine pH > 7.0, and severe struvite crystalluria. Presence of at least 2 of these signs was required for removal from study. Urine specimens were examined for electrolytes, struvite crystal content, and hematuria. RESULTS: Results for urine variables were compared between groups at 4 weeks, because of reduction in cat numbers attributable to removal from study. Struvite crystal content of 24-hour urine specimens was significantly lower for cats fed the choreito-containing diet. Moreover, frequency and severity of hematuria were significantly decreased in cats fed the choreito-containing diet. Correlation between hematuria and struvite crystal content was not observed in either group. Additionally, all 6 cats fed the diet without choreito had been removed from study by day 58 because of signs of LUTD. Of the 6 cats fed the choreito-containing diet, 2 completed the 12-week study. CLINICAL RELEVANCE: Choreito may be beneficial for relief of some signs of struvite-associated LUTD disease in cats.

Clinical evaluation of cats with nonobstructive urinary tract diseases.

OBJECTIVE: To identify the underlying cause of clinical signs in cats with nonobstructive diseases of the bladder and urethra. DESIGN: Prospective case series. SAMPLE POPULATION: 109 cats examined by the urology service of The Ohio State University's veterinary teaching hospital because of stranguria, hematuria, pollakiuria, or urination in inappropriate locations. PROCEDURE: History was obtained and a CBC, serum biochemical analyses, serologic tests for FeLV and feline immunodeficiency virus, urinalysis, bacterial culture of urine, and contrast radiography or urethrocystoscopy (females only) were performed. RESULTS: 16 cats had cystic calculi: 8 had struvite uroliths, 7 had calcium oxalate uroliths, and 1 had a urolith of unknown composition in conjunction with an anatomic defect. Anatomic defects, including diverticulae, urethral strictures, and a malpositioned urethra, were identified in 12 cats. A urinary tract infection was identified in 1 cat, and neoplasia was diagnosed in 2. One of the cats with neoplasia also had a struvite urolith. The remaining 80 cats did not have an anatomic defect, urolith, or tumor. Ten of these cats also did not have radiographic or cystoscopic abnormalities and were presumed to have a behavioral disorder. The remaining 70 cats had radiographic or cystoscopic abnormalities, and idiopathic cystitis was diagnosed. In 14 of the cats with idiopathic cystitis, results of a urinalysis were normal. Cats with idiopathic cystitis were significantly more likely to eat dry food exclusively (59%) than were cats in the general population (19%). CLINICAL IMPLICATIONS: Results suggest that idiopathic cystitis occurs commonly in cats with stranguria, hematuria, pollakiuria, or inappropriate elimination and is associated with consumption of dry foods. Contrast radiography or cystoscopy is necessary for differentiating idiopathic cystitis from behavioral disorders in some cats.

Base excision repair processing of radiation-induced clustered DNA lesions.

Energy from low LET ionising radiation, such as X rays and gamma rays, is deposited in the water surrounding the DNA molecule such that between 2 to 5 radical pairs are generated within a radius of I to 4 nm. As a result, multiple single lesions, including oxidised purine or pyrimidine bases, sites of base loss, and single-strand breaks, can be formed in DNA from the same radiation energy deposition event. The single lesions in these so-called multiply damaged sites or clustered lesions are repaired by base excision repair. Here we show that clustered DNA damages are formed in bacterial cells by ionising radiation and are converted to lethal double-strand breaks during attempted repair. In wild type cells possessing the oxidative DNA glycosylases that recognise and cleave DNA at repairable single damages, double-strand breaks are formed at radiation-induced clusters during post-irradiation incubation and in a dose-dependent fashion. Mutant cells lacking these enzymes do not form double-strand breaks post-irradiation and are substantially more radioresistant than wild type cells. These radioresistant mutant cells can be made radiosensitive by overexpressing one of the oxidative DNA glycosylases. Thus the effect of the oxidative DNA glycosylases in potentiating DNA damage must be considered when estimating radiation risk.

Abortive base-excision repair of radiation-induced clustered DNA lesions in Escherichia coli.

It has been postulated that ionizing radiation produces a unique form of cellular DNA damage called "clustered damages" or "multiply damaged sites". Here, we show that clustered DNA damages are indeed formed in Escherichia coli by ionizing radiation and are converted to lethal double-strand breaks during attempted base-excision repair. In wild-type cells possessing the oxidative DNA glycosylases that cleave DNA at repairable single damages, double-strand breaks are formed at radiation-induced clusters during postirradiation incubation and also in a dose-dependent fashion. E. coli mutants lacking these enzymes do not form double-strand breaks postirradiation and are substantially more radioresistant than wild-type cells. Furthermore, overproduction of one of the oxidative DNA glycosylases in mutant cells confers a radiosensitive phenotype and an increase in the number of double-strand breaks. Thus, the effect of the oxidative DNA glycosylases in potentiating DNA damage must be considered when estimating radiation risk.