Food additives: Sodium benzoate, potassium sorbate, azorubine, and tartrazine modify the expression of NFκB, GADD45α, and MAPK8 genes.

It has been reported that some of the food additives may cause sensitization, inflammation of tissues, and potentially risk factors in the development of several chronic diseases. Thus, we hypothesized that expressions of common inflammatory molecules - known to be involved in the development of various inflammatory conditions and cancers - are affected by these food additives. We investigated the effects of commonly used food preservatives and artificial food colorants based on the expressions of NFκB, GADD45α, and MAPK8 (JNK1) from the tissues of liver. RNA was isolated based on Trizol protocol and the activation levels were compared between the treated and the control groups. Tartrazine alone could elicit effects on the expressions of NFκB (p = 0.013) and MAPK8 (p = 0.022). Azorubine also resulted in apoptosis according to MAPK8 expression (p = 0.009). Preservatives were anti-apoptotic in high dose. Sodium benzoate (from low to high doses) dose-dependently silenced MAPK8 expression (p = 0.004 to p = 0.002). Addition of the two preservatives together elicited significantly greater expression of MAPK8 at half-fold dose (p = 0.002) and at fivefold dose (p = 0.008). This study suggests that some of the food preservatives and colorants can contribute to the activation of inflammatory pathways.

Expression pattern of markers in the canine ovarian cycle.

Although hormonal changes during different phases of the oestrous cycle of bitches are well-described, knowledge about the luteal phase and anoestrus is incomplete. Furthermore, which paracrine and autocrine critical factors that differentiate between follicles destined for atresia and those that continue to develop are unknown. In this study, ovarian tissue was collected from 39 healthy bitches that were subject to ovariectomy or ovariohysterectomy for surgical neutering or medical purposes such as unwanted pregnancy. Bitches were allocated to different groups depending on the stage of the oestrous cycle. Serum progesterone, LH, FSH and 17beta-estradiol (E(2)) -levels were determined and immunhistochemistry was performed for a variety of receptor antigens; Ki-67, vimentin, pan cytokeratin antibody, p53 and oestrogen receptor (ER) alpha antigens. Marked differences were found in progesterone concentration between pregnant and non-pregnant animals. Oestrogen concentration was significantly lower in pro-oestrus and ovulation than during the luteal phase. Although progesterone could be detected in cytoplasm of ovarian cells at each stage, its presence was restricted to follicular cells during anoestrus. A strong presence of AE1/AE3, vimentin and p53 was found in each oestrous stage, in contrast with Ki67. The localization of ERalpha appeared to vary during the oestrous cycle, a phenomenon that suggests a switch between target cells of oestrogen; while as a proliferation marker, the mild reaction of p53 during parturition suggests an apoptotic process at this stage of the cycle.

Progesterone, oestradiol, FSH and LH concentrations in serum of progesterone-treated pregnant bitches with suspected luteal insufficiency.

In the bitch, the corpus luteum is the only source of circulating progesterone throughout pregnancy. Inadequate luteal function may be a cause of abortion or foetal resorption observed after early pregnancy diagnosis. In our study of factors involved in canine luteal inadequacy, 35 pregnant bitches from different breeds were allocated to groups of healthy control (n = 15) vs hypoluteoid (n = 20) pregnant bitches, based on presence or absence of clinical signs of impending abortion and on progesterone concentrations below 10 ng/ml at the 4th-5th week of pregnancy. Hypoluteoid bitches were treated daily with 10-mg natural progesterone in injectable form (Luteosan inj.; Alvetra and Werfft AG, Vienna, Austria) until day 60. Serum progesterone, 17beta-oestradiol, FSH and LH concentrations were measured in samples obtained weekly using ELISA previously validated for dog serum. The exogenous progesterone supplementation was presumed to be sufficient to prevent foetal loss in 15 of the 20 treated bitches. The mean serum progesterone concentration in control pregnant bitches did not decrease below 10 ng/ml until the 8th week of pregnancy. Progesterone concentrations in progesterone-treated hypoluteoid bitches at week 4 were lower than in controls (p < 0.05), but although numerically higher did not differ significantly from those of controls during the period of treatment. Serum 17beta-oestradiol concentrations of healthy bitches were variable, were at most time higher than those of treated animals and slowly decreased until parturition; those of treated bitches remained unchanged during the study. Serum FSH and LH concentrations did not differ between groups. Additional studies involving untreated pregnancies showing equivalent evidence of hypoluteoidism as well as assay of circulating relaxin and prolactin in treated and untreated bitches are needed to better determine the causes and effects of hypoluteoidism in pregnant bitches.

Immunohistochemical detection of progesterone and cellular proliferation in canine mammary tumours.

Immunohistochemical expression of progesterone and the cellular proliferation marker Ki-67 was assessed in formalin-fixed, paraffin wax-embedded samples from 43 canine mammary tumours. Benign tumours showed high surface membrane progesterone expression (mean 196.42+/-25.91 positively labelled cells per 500 tumour cells) and low cellular proliferation (52.14+/-16.73 positively labelled cells per 500 tumour cells), whilst malignant tumours had low progesterone expression (68.19+/-17.53 positively labelled cells per 500 tumour cells) and higher cellular proliferation (141.72+/-23.65 positively labelled cells per 500 tumour cells), the difference being statistically significant (P<0.005) in both cases. These findings suggest that the majority of progesterone receptors in canine mammary tumour tissue are not associated with bound progesterone. The progression towards malignancy in spontaneously arising canine mammary tumours therefore appears to be associated with a decrease in steroid hormone dependency. Progesterone expression was also noted in the cytoplasm of tumour cells where it may be associated with a cellular repair mechanism. This hypothesis is supported by the finding of significantly higher progesterone content in the cytoplasm of benign tumour cells.

Effect of anticoagulants and sampling time on results of progesterone determination in canine blood samples.

The concentration of plasma progesterone was measured by ELISA, in serum and samples prepared with three different anticoagulant agents - namely ethylenediaminetetraacetic acid (EDTA), heparine and sodium fluoride oxalate potassium(NaFK). Forty clinically healthy bitches were selected based on the signs of pro-oestrus or oestrus. Values of progesterone concentration were significantly higher in serum than in EDTA-plasma (p < 0.0005); heparin-plasma (p < 0.05) and NaFK-plasma (p < 0.005). During pro-oestrus and oestrus until the time of ovulation, progesterone exhibited a conspicuous and statistically verified diurnal pattern (p < 0.05), its serum concentration being higher during 6.00-7.00 p.m. than 8.00-9.00 a.m. By the time of ovulation tendency of higher p.m. progesterone level reverses and from this point on the a.m. progesterone concentration is higher. The results of these experiments indicate that the concentration of canine progesterone assayed with ELISA may be affected by the time of collection and the method of preservation used.