Irina Pollard

Plasma glucocorticoid elevation and desynchronization of the estrous cycle following unpredictable stress in the rat.

Exposure of female Wistar rats to a signalled unpredictable 5-day stress regimen induced extreme plasma corticosterone elevation at all four stages of the estrous cycle. The corticosterone level at proestrus was significantly higher than at the other three stages of the cycle in both control and stressed groups. This rise in plasma corticosterone became evident only in late proestrus. It was observed that the estrous cycles of 50% of the rats became desynchronized over the 5-day stress period; a steroid hormone imbalance is suggested as a possible factor contributing to this desynchrony.

Accumulation of theophylline, theobromine and paraxanthine in the fetal rat brain following a single oral dose of caffeine

This paper describes the disposition of caffeine and its metabolites, theophylline, theobromine and paraxanthine in the 20-day fetal and adult brains following a single maternal dose of 5 or 25 mg/kg caffeine. Brains and plasma were collected 5 and 30 min, and 1, 3, 8 and 24 h after dosing. It was found that fetal and adult caffeine AUC (area under the concentration-time curve) values did not differ between the brain and plasma at either dose. Caffeines primary metabolites theophylline, theobromine and paraxanthine did, however, accumulate in the fetal brain at both doses resulting in a 3-fold increase in brain metabolite exposure compared to fetal circulatory levels. In contrast to the fetus, metabolite AUC values after a dose of 25 mg/kg were found to be lower in the brains of adults compared with plasma. This suggests that caffeines primary metabolites might be selectively excluded from the adult brain. In conclusion we have shown that, unlike the adult, the fetal rat brain accumulates theophylline, theobromine and paraxanthine when exposed to caffeine doses comparable to those attainable by normal human consumption. Since many aspects of caffeine metabolism are similar in the rat and human, we suggested that particular attention should be paid to the consumption of caffeine during pregnancy.

Plasma Glucocorticoid Elevation and Ultrastructural Changes in the Adenohypophysis of the Male Rat Following Prolonged Exposure to Stress

A morphological and ultrastructural study is described which indicates that cellular activity in the adenohypophysis correlates well with the circulating levels of corticosterone. Intense secretory activity is observed in all tropic cells of the adenohypophysis over 10 days; thereafter the cellular morphology shows a return to the control condition. There are, however, differences in the degree of adaptation between the different tropic cells. After its initial hyperactivity, corticotrope activity returned to a control level by 20 days. Thyrotrope activity was also found to adapt to control activity, but only after 40 days. Similar patterns were observed in the stomatotrope and gonadotrope, where the initial hypertrophy returned to control levels by 20 days; thereafter, however, an inhibition was observed. The luteotrope however, seems to be an exception in that its level of activity increased throughout the duration of the stress procedure.

Effects of caffeine administered during pregnancy on fetal development and subsequent function in the adult rat: prolonged effects on a second generation.

Caffeine, when administered in moderate (30 mg/kg X d) or high (60 mg/kg X d) doses during pregnancy, was shown to cause significant fetal growth retardation of both sexes. Mortality rate at or soon after birth was significantly higher and litter size significantly lower in the litters treated with 60 mg. The subsequent growth rates were also affected. The experimental pups grew more slowly, with growth plateauing at the same age resulting in smaller adults. The male offspring when subjected to short-term stress (one session) in adulthood showed an intact emergency response, demonstrating an adequate ability to react to a sudden environmental change. A significant decrease in 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity, and consequent reduction in testosterone biosynthesis, in the fetal testes at d 18 and 20 of gestation was also found for both doses of caffeine. Low 3 beta-HSD activity persisted to adulthood in the group receiving 60 mg. Lingering effects were observed in a second litter bred 8 wk after the discontinuation of caffeine consumption. In this second breeding, the offspring of both sexes from both caffeine doses were born significantly smaller when compared to the controls. Persistent effects of caffeine were also found in second-generation rats bred from females who were exposed to caffeine in utero. The pups of both sexes were born significantly heavier after a significantly longer gestation. The subsequent growth did not differ from that of the controls. It was suggested that a changed genetic program in the ovarian germ cells of the first generation and/or a changed uterine environment in the second generation may be implicated.

Caffeine exposure in utero increases the incidence of apnea in adult rats

Caffeine abuse during pregnancy may be a factor in the development of long-term breathing abnormalities. Therefore, the objective of the present study was to monitor adult breathing patterns after in utero exposure to caffeine. This was done by isolating episodes of apnea of more than 6-s duration from the breathing data as obtained by the Cotwatch breathing monitors adapted for rat use. The breathing record obtained over 6 consecutive days was expressed as daily weighted apnea-hypopnea density (WAHD) values. It was shown that administration of caffeine in moderate (30 mg/kg daily) or high (60 mg/kg daily) doses throughout gestation resulted in a significant dose-dependent increase in the WAHD value. The experimental offspring were significantly growth retarded in utero and their subsequent growth rates were also affected. The caffeine-exposed pups grew more slowly with growth plateauing at the same age, resulting in smaller adults. A link was suggested between infants with apnea of prematurity, when occurring after the first week, and an increased risk for later apnea and sudden infant death syndrome.

Caffeine-mediated effects on reproductive health over two generations in rats

The present study was designed to investigate the mechanism(s) underlying previously observed birth weight differences found in the first litter of the second (F2) generation bred from caffeine-exposed F1 females. The effect of exposure to caffeine in utero on subsequent sexual receptivity, fertility, gestation length, parturition, nesting activity, maternal behaviour, and reproductive senescence in the F1 mothers, and the viability of the F2 offspring was investigated. This information was collected by breeding control or caffeine exposed females for 8 consecutive litters. It was demonstrated that exposure to caffeine did not affect the sexual receptivity, fertility, gestation length, or maternal behaviour of the F1 females, but parturition was prolonged and the viability of the F2 generation was seriously jeopardized. Many F2 pups were born significantly larger than their control counterparts and a significant proportion of litters (after the first two litters) were wholly stillborn. It was concluded that a changed genetic program, mediated via the F2 fetus, delayed the normal progression of parturition. This, in turn, compromised the F1 mothers and caused increased mortality of their offspring. The severity of the outcome was dose dependent.

Effects of caffeine and its reactive metabolites theophylline and theobromine on the differentiating testis.

A previous study in the rat (Pollard et al. 1990) established that caffeine, when administered during pregnancy, significantly inhibited the differentiation of the seminiferous cords and subsequent Leydig cell development in the interstitium. However, that study could not distinguish between the direct effects of caffeine and/or the intermediary secondary toxic effects of metabolites such as theophylline and theobromine. Because the fetus lacks the appropriate enzyme systems, clearance of toxic substances takes place via the placenta and maternal liver. Thus, a suitable in vitro system can effectively differentiate between primary and secondary drug effects. In the present study, 13-day-old fetal testis, at the stage of incipient differentiation, were cultured for 4 days in vitro in the presence of graded doses of caffeine, theophylline or theobromine. It was found that explants exposed to caffeine or theobromine differentiated normally, developing seminiferous cords made up of Sertoli and germ cells, soon followed by the differentiation of functionally active Leydig cells appearing in the newly formed interstitium. However, explants exposed to theophylline failed to develop seminiferous cords and, as a consequence, Leydig cells. In conclusion, insights obtained from different experimental methods, such as organ culture or whole organism studies, are not always identical. It may be prudent, therefore, to take into account that certain experimental techniques, despite providing valuable information, may require confirmation by other test methods in order to obtain an in-depth understanding of mechanisms of action involved.

Effects of preconceptual caffeine exposure on pregnancy and progeny viability.

OBJECTIVE A previous study demonstrated for the first time that a drug such as caffeine, administered prior to ovulation and genomic activation, causes a quantitative difference in growth-promoting energy utilization in a proportion of 5-day-old blastocysts. The objective of the present study was to investigate whether developmental changes induced by caffeine administered throughout the estrus cycle prior to fertilization are sustained throughout pregnancy and after birth. METHODS Caffeine was administered to rats throughout the estrus cycle prior to fertilization, with control and experimental groups subdivided into preimplantation and postimplantation categories. Preimplantation fertilization rate was assessed on day 4 of pregnancy by a pregnancy-induced elevation in maternal plasma progesterone concentration, or by flushing each uterine horn on day 5 of pregnancy to determine the presence or absence of a litter. Postimplantation fetuses were collected on gestational day 12 or allowed to go to term. RESULTS Preconceptual caffeine exposure significantly reduced maternal fertility by the failure of a proportion of the litters to implant, rather than curtailing preimplantation development or postimplantation losses. Postnatal mortality between weeks 0 and 1 was elevated and the weekly incremental growth rate of the pups from week 3 through week 7 was significantly reduced in the preconceptually caffeine-treated offspring. Experimental females reached puberty at the same age as the controls but at a significantly lower body weight. Gestation length, hirthweight, litter size, sex ratio, and anogenital distance (a measure of prenatal androgenization) were not affected by preconceptual caffeine treatment. CONCLUSIONS It was concluded that the reduced fertility rate in preconceptually caffeine-exposed rats was due to the failure of litters to implant rather than to a reduced fertilization rate, which was normal. It was further concluded that the growth rate over the neonatal and prepubertal periods of surviving pups in the caffeine-treated group was subnormal.

Immunohistochemical localisation of the 90, 70 and 25 kDa heat shock proteins in control and caffeine treated rat embryos.

Human and animal experimental data demonstrate that in utero exposure to caffeine results in intrauterine growth retardation and long-term behavioural and reproductive effects. We have suggested that the disruption of normal transcription and translation associated with the initiation of the heat shock response may be a possible mechanism of action of caffeine. This hypothesis was investigated using immunohistochemistry to determine whether an acute (3 h) dose of 30 mg/kg caffeine alters the distribution of hsp 90, 70 and 25 in 10.5-12.5 g.d. rat embryos. In the control embryos hsps 90 and 70 were distributed throughout the embryo with no areas of specific accumulation. Hsp 25 was localised to the developing myocardium of 10.5, 11.5 and 12.5 g.d. embryos and the myotome of 11.5 and 12.5 g.d. embryos. The appearance of hsp 25 was correlated with the onset of muscle fibre differentiation and it is suggested that hsp 25 is associated with cytoskeletal proteins. Following dosing with caffeine no change in the distribution of staining for hsp 90, 70 and 25 was found. These results strongly suggest that caffeines mechanism of action does not involve initiation of the heat shock response.

Caffeine does not increase synthesis of heat shock proteins in rat embryos

Caffeine exposure in utero in rats is known to result in intrauterine growth retardation and lowered birth weight as well as changes to behaviour and brain biochemistry. We have investigated whether caffeines embryotoxicity is a result of the events associated with increased hsp synthesis, i.e., disruption to normal protein synthesis. Caffeine (30 mg/kg) was administered orally to pregnant rats as single or repeated doses. Embryos were removed 3 h after dosing on gestation day (GD) 9, 10, 11 and 12 and total embryonic protein and RNA analysed. There was no change in the mRNA or protein levels of hsp 88, 71/73, and 25 after acute or chronic treatment. To separate the direct effect of caffeine from those mediated through the mother, whole rat embryo culture was used. Caffeine (50 micrograms/ml) for 90 min did not increase hsp 88, 73 or 25 mRNA levels in 9.5, 10.5 and 11.5 GD cultured embryos. We conclude that in vivo or in vitro treatment of 9-12 GD rat embryos with moderate to high doses of caffeine does not increase the synthesis of the major mammalian hsps. Hence, hsp induction is unlikely to play a role in the embryotoxic actions of caffeine.