Simultaneous and sensitive determination of xanthotoxin,psoralen, isoimpinellin and bergapten in rat plasma by liquid chromatography–electrospray ionization mass spectrometry

A sensitive, specific and rapid liquid chromatography–mass spectrometry (LC–MS) method has been developed and validated for the simultaneous determination of xanthotoxin (8-methoxypsoralen), psoralen, isoimpinellin (5,8-dimethoxypsoralen) and bergapten (5-methoxypsoralen) in rat plasma using pimpinellin as an internal standard (IS). The plasma samples were pretreated by protein precipitation with methanol and chromatographic separation was performed on a C₁₈ column with a mobile phase composed of 1 mmol ammonium acetate and methanol (30:70, v/v). The detection was accomplished by multiple-reaction monitoring (MRM) scanning via electrospray ionization (ESI) source operating in the positive ionization mode. The optimized mass transition ion-pairs (m/z) for quantitation were 217.1/202.1 for xanthotoxin, 187.1/131.1 for psoralen, 247.1/217.0 for isoimpinellin, 217.1/202.1 for bergapten, and 247.1/231.1 for IS. The total run time was 6 min between injections. The calibration curves were linear over the investigated concentration range with all correlation coefficients higher than 0.998. The lower limits of quantitation (LLOQ) of these analytes were less than 1.21 ng/ml. The intra- and inter-day RSD were no more than 9.7% and the relative errors were within the range of ?8.1% to 4.5%. The average extraction recoveries for all compounds were between 90.7% and 106.2%. The proposed method was further applied to the determination of actual plasma samples from rats after oral administration of Radix Glehniae extract.     

Accumulation of furanocoumarins in Ruta graveolens L. shoot culture

Ruta graveolens L. shoots cultured in stationary liquid phase produced furanocoumarins: psoralen, bergapten, xanthotoxin, isopimpinellin and imperatorin at the amount totalling almost 1 g/100 g dry wt of the shoots. The dominating metabolites were therapeutically important compounds: xanthotoxin – 0.33 g/100 g dry wt and bergapten – 0.32 g/100 g dry wt. Maximum contents of the majority of the compounds were observed on 28th day of culture.     

Plasma corticosterone level of rats after bilateral lesion of the globus pallidus and elimination of the sex-dependence of the body weight loss following pallidal lesion by bilateral adrenalectomy

Plasma corticosterone (CST) level and body weight were studied in male and female rats after bilateral pallidal lesion (GPL). The effect of high dose of glycocorticoids on body weight of food and water deprived, non-lesioned rats was also studied since high CST levels due to the stress caused by the aphagia and adipsia could be anticipated. Weight changes in lesioned-adrenalectomized animals were in investigated as well. A weight loss, higher in males than females, developed after the pallidal lesion and the CST level was higher in lesioned females than in lesioned males. This sex-difference was present in non-lesioned, food and water deprived animals as well. The weight loss was higher in lesioned males than in food and water deprived male/female controls. Adrenalectomy eliminated weight loss differences between the sexes after lesion, by reducing the weight loss in males. CST administration reduced body weight loss in food and water deprived males but not in familes, and cortisol facilitated it in both sexes.     

Maternal Deprivation Exacerbates the Response to a High Fat Diet in a Sexually Dimorphic Manner

Maternal deprivation (MD) during neonatal life has diverse long-term effects, including affectation of metabolism. Indeed, MD for 24 hours during the neonatal period reduces body weight throughout life when the animals are maintained on a normal diet. However, little information is available regarding how this early stress affects the response to increased metabolic challenges during postnatal life. We hypothesized that MD modifies the response to a high fat diet (HFD) and that this response differs between males and females. To address this question, both male and female Wistar rats were maternally deprived for 24 hours starting on the morning of postnatal day (PND) 9. Upon weaning on PND22 half of each group received a control diet (CD) and the other half HFD. MD rats of both sexes had significantly reduced accumulated food intake and weight gain compared to controls when raised on the CD. In contrast, when maintained on a HFD energy intake and weight gain did not differ between control and MD rats of either sex. However, high fat intake induced hyperleptinemia in MD rats as early as PND35, but not until PND85 in control males and control females did not become hyperleptinemic on the HFD even at PND102. High fat intake stimulated hypothalamic inflammatory markers in both male and female rats that had been exposed to MD, but not in controls. Reduced insulin sensitivity was observed only in MD males on the HFD. These results indicate that MD modifies the metabolic response to HFD intake, with this response being different between males and females. Thus, the development of obesity and secondary complications in response to high fat intake depends on numerous factors.     

Gender difference in serum bisphenol A levels may be caused by liver UDP-glucuronosyltransferase activity in rats

Gender difference in human bisphenol A (BPA) concentrations was revealed by determining serum BPA. We studied the serum concentrations and the metabolism of BPA in rats by an HPLC system. Rat serum BPA concentrations were significantly higher in males (24.9+/-7.38 ng/ml, P=0.026, n=10) than in females (8.27+/-3.11 ng/ml, n=10), as in humans. The resultant enzyme reaction products of BPA glucuronidation in the rat liver microsomes fraction were analyzed by an HPLC system. The ratio of BPA glucuronidation in the microsome reaction was significantly higher (P=0.015) in female than in male rats. The mRNA expression of UDP-glucuronosyltransferase 2B1 (UGT2B1), an isoform of UGT related to BPA glucuronidation, in the rat liver was analyzed by a real-time quantitative RT-PCR. The relative expression level of UGT2B1 mRNA was significantly higher (P<0.001) in female than in male rat livers. The gender difference in serum BPA concentrations may be explained by the difference in clearance based on the UGT activities.     

Influence of gender on tamoxifen-induced biochemical changes in serum of rats

Tamoxifen, the widely prescribed drug in the prevention and therapy of breast cancer, may cause side effects which may be influenced by gender. The present study was undertaken to investigate the impact of gender on tamoxifen-induced toxic and biochemical changes following oral administration of tamoxifen at high dose level of 20 mg/kg once daily for a 2-week period in both male and female rats. The results showed marked increases in serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in female rats. In contrast, treatment with tamoxifen in male animals significantly decreased the activity of ALT, with a tendency for a decrease in serum AST levels. In female rats, a significant reduction in the serum activity of acid phosphatase (ACP) was noted, compared with a non-significant decrease in males. Non-significant changes in serum levels of alkaline phosphatase (ALP) were seen in both sexes. Tamoxifen lowered serum contents of total lipid and total cholesterol in both male and female rats. Serum levels of triglycerides were reduced in female rats as compared to a non-significant decrease in male animals. The serum albumin concentration was decreased in both male and female rats, while total protein was decreased only in female animals. Tamoxifen markedly increased serum levels of creatinine in female rats, compared with a non-significant rise in males. Total serum contents of calcium were similarly reduced in both males and females. This is the first study which points to gender-related differences in tamoxifen-induced toxic and metabolic changes in rats. The results indicated that females are more susceptible than males to tamoxifen toxicity, probably due to the ability of tamoxifen to antagonize the action of estrogen in females.     

Furanocoumarin metabolism in Papilio polyxenes: biochemistry,genetic variability,and ecological significance

The ubiquitous occurrence of series of biosynthetically related plant secondary compounds within individual species has given rise to the suggestion that such multiplicity is adaptive; one possible mechanism that would serve to maintain such within-plant diversity is analog synergism. In a series of experiments, we provide evidence that synergism may account for the presence of multiple structurally related furanocoumarins in apiaceous plants. The black swallowtail, Papilio polyxenes, feeds exclusively on plant species containing furanocoumarins. Growth of larvae fed parsley leaves treated with both xanthotoxin and angelicin, two furanocoumarins that co-occur widely in swallowtail hostplants, was significantly slower than that of larvae fed leaves with an equimolar concentration of either xanthotoxin or angelicin. A multivariate combination of growth, food consumption and frass excretion differed significantly between larvae fed leaves treated with both xanthotoxin and angelicin and larvae fed leaves treated with angelicin alone. In addition, we measured rates of in vitro cytochrome P450-mediated metabolism of three furanocoumarins — bergapten, xanthotoxin, and angelicin. While bergapten and xanthotoxin, both linear furanocoumarins, were metabolized at similar rates (8.07 and 9.86 nmoles/min/g fw caterpillar, respectively), angelicin, an angular furanocoumarin, was metabolized more slowly (2.76 nmoles/min/g fw caterpillar). When all three furanocoumarins were assayed together, overall rates of metabolism were significantly reduced, suggesting substrate inhibition. Thus, the pattern of growth of larvae is consistent with the pattern of in vitro metabolism and is evidence in support of analog synergism. In a separate experiment, metabolism of xanthotoxin and angelicin individually and together were compared in six maternal families. Again, angelicin was metabolized more slowly than xanthotoxin and each furanocoumarin inhibited metabolism of the other. That significant family effects were found for rates of metabolism and for the ratio of moles of angelicin metabolized for each mole of xanthotoxin metabolized raises the possibility that genetic variation exists for the rate and specificity of metabolism and suggests that insect herbivores may be able to adapt to analog synergism.     

Gender related alterations of β-adrenoceptor mechanisms in heart failure due to arteriovenous fistula

This study was undertaken to determine gender related changes in different components of β-adrenoceptor (β-AR) system in response to arteriovenous fistula (AV-shunt), which is known to produce heart failure due to volume overload. AV-shunt was induced in male and female rats for 16 weeks by the needle technique; ovariectomized (OVX) rats treated with or without estrogen were also used. Although AV-shunt for 16 weeks produced cardiac hypertrophy in both sexes, male animals showed cardiac dysfunction whereas cardiac performance was maintained in females. Both β(1) -AR and β(2) -AR protein content and mRNA levels were decreased in male and increased in female hearts post-AV-shunt. The basal adenylyl cyclase (AC) activity was lower in the female heart; however, AC protein content and the increase in epinephrine (EPi)-stimulated AC activity were greater in the female AV-shunt group as compared to males. While AC V/VI and β-arrestin 2 mRNA levels were decreased in males, mRNA level for GRK2 was increased in females post-AV-shunt. In contrast to intact females, AV-shunt OVX animals showed depressed cardiac function, decreased β(1) -AR, β(2) -AR, and AC protein content, as well as reduced EPi-stimulated AC activity. Treatment of OVX rats with 17-β estradiol attenuated the AV-shunt induced changes in β-AR and AC protein content as well as cardiac dysfunction. These results reveal that β-AR signal transduction system in response to AV-shunt is downregulated in males and upregulated in females. Furthermore, estrogen appears to play an important role in the upregulation of β-AR mechanisms and the maintenance of cardiac function in AV-shunt females.     

Puberty in pine voles, Microtus pinetorum, and the influence of chemosignals on female reproduction

We investigated the reproductive biology of an induced ovulator, the pine vole (Microtus pinetorum). Male puberty, measured as age at first impregnation, was found to occur as early as 44 days of age. Female puberty measured as age at first conception, was found to occur as early as 32 days of age, considerably earlier than previously reported. Females paired with stud males exhibited a doubling of uterine weight within 12 h, and vaginal sperm were present after 48 h. This indicates that although behavioral responses to males--including mating--require prolonged contact, physiological responses to males occur rapidly. Chemosignals from males slightly increased uterine and ovarian weights of females, but chemosignals from other females did not. Young females paired with stud males for 48 h in the presence of soiled bedding from the female's family had significantly smaller increases in ovarian and uterine weights than similar females paired on clean bedding. Suppression of reproduction in female offspring while they remain with the extended family unit is discussed as a life-history tactic and as a possible mechanism for inbreeding avoidance.     

Comparative effects of tamoxifen and angiotensin II type-1 receptor antagonist therapy on the hemodynamic profile of the ovariectomized female rat

Hormonal replacement therapy (HRT) has failed to provide a cardioprotective action in postmenopausal women, and thus alternative pharmacological approaches are required. The present study examined the therapeutic potential of the partial estrogen receptor agonist tamoxifen and the angiotensin II type-1 receptor antagonist irbesartan on the hemodynamic profile of ovariectomized (OVX) female Sprague-Dawley rats (9-11 weeks). Three weeks following ovariectomy, uterine atrophy was evident and body weight was increased as compared with sham-operated animals. Left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), and mean arterial pressure (MAP) were significantly increased in the OVX rats as compared with sham rats. One week following ovariectomy, rats were treated with either tamoxifen (10 mg kg-1 day-1) or irbesartan (40 mg kg-1 day-1) for a period of 2 weeks. The administration of tamoxifen to OVX rats partially reversed uterine atrophy and prevented body weight gain, albeit body weight remained significantly lower than in sham-operated animals. LVSP and LVEDP were normalized in the tamoxifen-treated OVX rats, whereas MAP remained elevated. Irbesartan partially reduced the body weight gain of the OVX rats and did not influence uterine atrophy. LVSP and MAP were normalized in irbesartan-treated OVX rats, whereas LVEDP remained elevated. These data demonstrate that irbesartan rather than tamoxifen was efficacious in normalizing MAP in the OVX rats without a secondary effect on the uterus.     

Effects Of A Post-Weaning Cafeteria Diet In Young Rats: Metabolic Syndrome,Reduced Activity And Low Anxiety-Like Behaviour

Among adolescents, overweight, obesity and metabolic syndrome are rapidly increasing in recent years as a consequence of unhealthy palatable diets. Animal models of diet-induced obesity have been developed, but little is known about the behavioural patterns produced by the consumption of such diets. The aim of the present study was to determine the behavioural and biochemical effects of a cafeteria diet fed to juvenile male and female rats, as well as to evaluate the possible recovery from these effects by administering standard feeding during the last week of the study. Two groups of male and female rats were fed with either a standard chow diet (ST) or a cafeteria (CAF) diet from weaning and for 8 weeks. A third group of males (CAF withdrawal) was fed with the CAF diet for 7 weeks and the ST in the 8th week. Both males and females developed metabolic syndrome as a consequence of the CAF feeding, showing overweight, higher adiposity and liver weight, increased plasma levels of glucose, insulin and triglycerides, as well as insulin resistance, in comparison with their respective controls. The CAF diet reduced motor activity in all behavioural tests, enhanced exploration, reduced anxiety-like behaviour and increased social interaction; this last effect was more pronounced in females than in males. When compared to animals only fed with a CAF diet, CAF withdrawal increased anxiety in the open field, slightly decreased body weight, and completely recovered the liver weight, insulin sensitivity and the standard levels of glucose, insulin and triglycerides in plasma. In conclusion, a CAF diet fed to young animals for 8 weeks induced obesity and metabolic syndrome, and produced robust behavioural changes in young adult rats, whereas CAF withdrawal in the last week modestly increased anxiety, reversed the metabolic alterations and partially reduced overweight.     

Bacteriological tests as an aid in the management of dental caries in Iceland

The activities of three natural coumarins, xanthotoxin and bergapten (fromAmmi majus, Umbelliferae) and psoralene (fromFicus cycomorus, Moraceae), were tested against mycelial growth and aflatoxin production of a toxigenic strain ofAspergillus flavus grown in a rice/corn steep liquor medium. Two other natural chromones, khellin and visnagin (fromAmmi visnaga) were also compared. Complete inhibition of aflatoxin release occurred with either xanthotoxin or khellin at 5 mM. The other three compounds also at 5 mM reduced aflatoxin to 12 to 16% of its original concentration. The mould growth was only slightly inhibited by all the compounds used.     

Age and sex dependence of the effects of an aqueous extract of Physalis alkekengi fruits on rat hepatic glucose 6-P dehydrogenase activity

Intraperitoneal injections of an aqueous extract of winter cherry fruits (Physalis alkekengi) to new-born, weanling and adult female rats and to weanling and adult male rats had no effect on body weight, liver weight and liver cytosol protein content. The specific activities of hepatic glucose 6-P dehydrogenase (an estrogen induced protein) in rats of different age and sex groups in terms of mU/mg protein were: treated new-born females, 15.9 ± 0.5; control, 29.1 ± 0.6; treated weanling females, 14.9 ± 0.3; control, 24.8 ± 0.7; treated adult females, 25.7 ± 0.5; control, 26.1 ± 0.5; treated weanling males, 7.9 ± 0.2; control, 7.9 ± 0.1; treated adult males, 9.6 ± 0.4; and control, 9.7 ± 0.3. Treatment of new-born and weanling female rats with the extract resulted in 40–45% reduction in hepatic G6PD activity. However, treatment of adult females, and weanling and adult males produced no significant change in the activity of this enzyme. The data are discussed both in terms of the increase in the capacity of rodent liver to metabolize steroidal compounds with age and the presence of low levels of circulating estradiol necessary for enzyme induction in male rats.     

Inhibition of thyroxine binding to adenohypophysial proteins in vitro by 2,4-dinitrophenol administered in vivo.

Male and female rats were given oestradiol benzoate (1 mg as a microcrystal aqueous suspension i.m. twice a week), 0.0033% 2.4-dinitrophenol (DNP) in their food (about 1 mg/rat/day), or 0.1% DNP in their food (about 30 mg/rat/day), or both oestradiol and DNP. The smaller DNP dose mildly stimulated food consumption and did not affect body weight. The larger dose strongly inhibited food consumption in the first two weeks of the experiment; consumption then returned to the control level, but body weight fell markedly at the same time. After 3 weeks' administration of both the small and the large dose of DNP, adrenal weight in the males was raised and the weight of the gonads was unchanged. The large DNP dose severely reduced the weight of the seminal vesicles and the uteri. It also inhibited the accumulation of radioiodine in the thyroid of both males and females. Isolated administration of the oestrogen raised adrenal weight in the males and ovarian and uterine weight in the females; it reduced the weight of the testes and seminal vesicles. These reactions were not affected by DNP. A pronounced oestradiol-induced increase in the weight of the adenohypophyses was accompanied by raised thyroxine binding to the adenohypophysial proteins in vitro. DNP inhibited the growth reaction of the adenohypophysis to the oestrogen only slightly and non-significantly, but significantly inhibited the thyroxine binding reaction to the adenohypophysial proteins in vitro. By itself, DNP had no effect on adenohypophysial weight, but reduced thyroxine binding to the adenohypophysial proteins in vitro, especially in males. The effect of DNP was similar to that of thyroxine observed in earlier experiments; nothing is known of its mechanism.     

Relationship between functional castration and alpha-fetoprotein produced by hepatoma-bearing female rats.

In rats bearing Morris hepatoma No. 7777, serum levels of alpha fetoprotein (AFP) increased as tumour size increased. Hepatoma-bearing females remained in dioestrus once the tumour exceeded a cross-sectional area of 12 cm2. The following changes were seen in tumour-bearing rats compared to control animals: uterine wet weight was significantly decreased, the uterine epithelial mucosa was lower, castration cells developed in the anterior pituitary, and there was a 3- to 23-fold elevation in serum oestrogen levels. We conclude that circulating oestrogen is bound to AFP in hepatoma-bearing female rats and that in this bound state it is unable to exert its normal physiological actions.     

Identification of Ruta graveolens L. metabolites accumulated in the presence of abiotic elicitors

The study aimed to elucidate the effects of benzothiadiazole (BTH) and saccharin on the biosynthesis of simple coumarins, linear furanocoumarins, dihydrofuranocoumarins, and furoquinolone alkaloids in shoots of R. graveolens cultivated in vitro. The biosynthesized metabolites were analyzed and identified by GC-MS and by comparison of Kovats indices. Eight coumarin metabolites were identified: bergapten, chalepin, isopimpinelin, pinnarin, psoralen, rutacultin, rutamarin, and xanthotoxin, and also four alkaloids: dictamnine, gamma-fagarine, skimmianine, and kokusaginine. Each of the tested BTH concentrations induced a significant production of furanocoumarins and furoquinolone alkaloids. The use of saccharin also increased the production of bergapten, isopimpinelin, pinnarin, psoralen, and xanthotoxin several times.     

Isolation and Identification of Xanthotoxin (8-Methoxypsoralen) and Bergapten (5-Methoxypsoralen) from Celery Infected with Sclerotinia sclerotiorum

Methods for the production, isolation, and identification of xanthotoxin and bergapten from celery diseased by Sclerotinia sclerotiorum (Libert) de Bary were investigated. The only conditions under which this mold was capable of producing xanthotoxin and bergapten occurred when the mold was actively growing on fresh (metabolizing) celery. Neither compound was found in uninfected celery, in the mold growing on nutrient media, on nutrient media fortified with 30% filter-sterilized celery juice, or on nonmetabolizing celery. Maximal xanthotoxin production of 320 mug per g of dry rotted celery occurred at 20 C, although mycelial growth increased until 30 C. Neither xanthotoxin nor bergapten was found when the mold grew on 11 agricultural commodities other than celery.