Cross-Species Similarities in Metabolism
One reason that animal models are useful in the study of caffeine is that the pharmacokinetics of caffeine may be similar to humans in some animal species. In both animals and humans, oral administration of caffeine results in its rapid absorption, with peak plasma levels attained within 3 to 120 min (Perves and Sullivan, 1993
). The absorption rates also increase with increased dosages in both humans and animals, and there is no significant first-pass effect, although absorption and the intestinal milieu do affect absorption, differing slightly in timing of distribution, but otherwise comparable in attained blood levels. Tomimatsu et al. (2007
) described caffeine as hydrophobic and rapidly passing through all biological membranes, including the blood–brain and placental barriers in sheep. Absorption from the gastrointestinal tract is rapid in adult humans, with attainment of maximum caffeine concentrations within 15 to 60 min after oral ingestion (for dosages of 5 to 8 mg/kg, the plasma concentrations equaling 8 to 10 µg/ml) Supplemental . Once absorption occurs, caffeine is rapidly distributed in body water, equilibrating between blood and tissues, including the embryo/fetus, as well as the brain and testes. It is also rapidly distributed to the breast milk. Caffeine in human breast milk contains approximately 75% of the plasma level, depending upon the maternal dosage (3.2–8.6 µg/ml of caffeine is found in human breast milk and 0.7–7.0 µg/ml in rat milk). Consumption of caffeine in the milk results in only 1% of the maternal intake being consumed by human infants and 2% of the maternal intake consumed by rat pups (Perves and Sullivan, 1993
Pregnancy alters the metabolism of caffeine, which, under normal conditions, is rapidly metabolically eliminated. Caffeine's retention is increased during pregnancy in humans, late human fetuses, and neonates, with a half-life varying from 80 to 100 hr. Presumably this increase in retention is the result of deficient P-450 enzymes in the fetus and neonate. Human metabolism of caffeine reaches adult parameters after approximately 7 months of age, but the half-life can be affected by inducing agents. For example, the half-life in smokers is approximately half of that in nonsmokers (Christian and Brent, 2001
The characterization of the enzymatic process of caffeine metabolism was also explored by Buters et al. (1996
), who investigated the involvement of CYP1A2 metabolizing enzymes in the pharmacokinetics and metabolism of caffeine using mice lacking its expression (CYP1A2−/−
). The mice were intraperitoneally administered 2 mg/kg of caffeine, a dosage that was reported to be equivalent to that of a human drinking one cup of coffee. The half-life of caffeine elimination from blood was seven times longer, AUC was increased eight times, and clearance was consequently eight times longer in these animals than in wild-type mice. Other P450 enzymes were not affected and the clinical pathology evaluations of the liver and kidney were unaffected. These data indicate that the clearance (elimination) of caffeine in wild-type mice is primarily determined by CYP1A2. Because human and mouse CYP1A2 resemble each other in cDNA-derived amino acid sequence, these data also suggest that humans have a similar elimination pattern.
Derkenne et al. (2005
) confirmed the conclusions of previous investigators that mouse or human CYP1A2 is the predominant enzyme for theophylline metabolism. Seven blood samples were taken at intervals from 5 to 400 min after IP injection of 8 mg/kg theophylline in mice. Replacing mouse CYP1A2 (− / −) with a functional human CYP1A2 gene restored the ability to metabolize theophylline, and the metabolism changed to a human profile. Comparing the hCYP1A1_A2 Cyp1a2 (− / −) and wild-type mice with published clinical studies revealed that theophylline clearance to be approximately 5 × and 12 ×, respectively, greater than that reported in humans, which is due to the well-known fact that mice clear drugs more rapidly than humans. Metabolism of caffeine varies remarkably among species and within the same species, and it is highly dependent on variables such as sex, age, and pregnancy status. In human newborns, the plasma half-life of caffeine is 4 days, while in young children and teenagers (6–13 years old), the plasma half-life is 2.3 hr. In adult humans, the half-life averages 2-6 hr in healthy nonsmokers, but it is prolonged in pregnant women to 10 to 20 hr. In rats, a half-life of 2.12 hr is reported for 8-week-old Sprague–Dawley male rats given one oral dosage of 4 mg/kg of caffeine. The major metabolite in humans is paraxanthine, or 1,7-dimethylxanthine. In rats, the major metabolite is 1, 3, 7-diaminouracil, or 6-amino-5-[N
-formylmethylaminol]-1,3-dimethyluracil. Caffeine is demethylated in both rats and humans to three dimethylxanthenes (theophylline, theobromine, and paraxanthine), which suggests that rats are an appropriate model for use in risk assessment for humans.
The differences in caffeine and paraxanthine metabolism between human and murine CYP1A2 in liver microsomes were also explored by Labedzki et al. (2002
). Results of the in vitro studies confirmed the important role of CYP1A2 in both murine and human metabolism of caffeine, despite formation of 1, 3, 7-trimethylurate as an in vitro “artifact” in both human and murine microsomal preparations. Both human and murine CYP1A2 enzymes have close similarities in the primary metabolic steps of caffeine. However, paraxanthine in vivo was not metabolized by murine CYP1A2 to a relevant extent, which is in contrast to the human situation. Also, results of this study confirmed the known reported inhibitory effects of the quinolones, norfloxacin, and pefloxacin on human CYP1A2, while in murine hepatic microsomes, quinolones did not exert an inhibition of caffeine 3-demethylation. The authors concluded that murine models are important for understanding the metabolism of xenobiotics in humans, but that extrapolation of data may be inaccurate in certain cases, such as in cases where compounds have low affinity ligands to CYP1A2. Therefore, interspecies comparison may be required before the use of mouse models to predict toxicity and/or pharmacological activity in humans. However, the metabolic patterns in rats are more closely related to the human.
Effect of Caffeine on the Neonate
The capability to adequately metabolize xenobiotics are greatly reduced in neonatal or premature infants and animals due to an inadequately developed hepatic enzyme system, and often it is difficult to determine exact medicinal dosages during this age. In humans, intravenous theophylline is frequently administered to premature neonates during the first several days to reduce apnea, although there has been little emphasis in the literature on the pharmacokinetics in this segment of the population. Two clinical trials on this subject are presented below to describe some of the pharmacokinetic parameters.
The clearance rate (CL) and volume of distribution (V) of theophylline were studied by du Preez et al. (1999
) in 105 apneic premature neonates (mean weight: 1.3 kg; age: 1.1 days) receiving intravenous loading dosages of 4 to 7.7 mg/kg aminophylline. Maintenance dosages ranged from 1.4 to 6 mg/kg/day in 2 to 4 divided doses. Data were analyzed using the nonlinear mixed effects model (NONMEM), and a one-compartment model with first-order elimination. The study differed from other cited premature neonatal references in that it was conducted in South Africa on all-black babies that had a 92% incidence of respiratory distress syndrome, and the described PK related only to the first few postnatal days. Low CL values were recorded (0.0084 and 0.056 l/hr/kg, respectively, for babies with and without oxygen support), while values of ≥0.012 l/hr/kg have been cited by other investigators. As a result of the low CL, long half-lives (54 and 76 hr, respectively, for babies with and without oxygen support) were reported. The calculated value for V was 0.63 l/kg. Variability in both CL and V were high, and it was concluded that theophylline PK is highly variable in neonates because physiologic parameters are changing rapidly and theophylline clearance and urinary metabolite patterns apparently do not reach stable adult values until 55 weeks postconception.
Urinary output was also evaluated in 19 premature infants aged 4.5 ± 4.0 days before and after a 20-min loading solution of aminophylline (6 mg/kg), which was followed by a maintenance therapy of 2 mg/kg every 12 hr (Mazkereth et al., 1997
). The infants had a mean gestational age of 31.1 ± 2.8 weeks and a birth weight of 1481 ± 454 g. Marked diuresis occurred immediately after the loading dose, and the ratio of urinary output to water intake increased from 0.58 ± 0.36 to 1.19 ± 0.65. Fractional excretion of sodium and potassium increased, and urinary calcium and uric acid excretion was also enhanced. Tubular reabsorption of phosphorus was not affected. These effects were no longer evident after 24 hr, despite aminophylline maintenance therapy. The authors concluded that the aminophylline acted directly on tubular reabsorptive functions of the nephron. Neonatal patients afflicted with hyperbilirubinemia may also gain some benefit from a neonatal rat model that could be used to evaluate new therapeutic agents for this disease. Induction of cytochrome P450 1A (CYP1A) may be a valuable therapeutic modality for reducing the hyperbilirubinemia of infants with Crigler–Najjar syndrome type I (CNS-I), a severe form of congenital jaundice. To evaluate inducers of CYP1A, a novel assay was established by Jorritsma et al. (2000
), based on the comparison of the type of urinary pattern of caffeine metabolites in rats when 10 mg/kg of 1-Me-14C-caffeine is injected intraperitoneally before and 48 hr after injection of a potential CYP1A inducer, such as 5,6-benzoflavone (BNF). The inducing effect of BNF on CYP1A activity was confirmed by the urinary pattern of caffeine metabolites in Wistar rats and was paralleled by a decrease in plasma bilirubin in male jj
It is interesting to note that in conjunction with the above study, a selective and sensitive reverse-phase liquid chromatographic method was developed by Schrader et al. (1999
) for the simultaneous analysis of [1-methyl-14C] caffeine and its eight major radiolabel-led metabolites in rat urine. Separation of the complex mixture of metabolites was achieved by gradient elution with a dual solvent system using an endcapped C18 reverse-phase column, which, in contrast to commonly used C18 reverse-phase columns, also allows the separation of the two isomers of 6-amino-5-(N
-formylmethylamino)-1,3-dimethyluracil (1,3,7-DAU), a metabolite of quantitative importance predominantly occurring in rats.
Impact of Various Factors on Altering the Pharmacokinetics of Caffeine
The effect of gender on the pharmacokinetics of caffeine (5 mg/kg, intravenously) was explored in 10 male and 10 female Holstein cattle during the ages 1, 2, 4, 6, 8, 12, and 18 months (Janus and Antoszek, 2000
). The findings were compared to the results in other species, including humans. The volume of distribution (V
) decreased significantly with age, as it does in pigs and humans; results were similar in males and females. A steady, significant decrease in mean residence time (MRT) also occurred in both sexes, although the MRT was significantly shorter in females after 8 months of age. Significant decreases over time also occur in dogs, pigs, and humans because caffeine clearance depends principally on intrinsic hepatic clearance. Total plasma clearance (Cl) of caffeine increased by nearly 100% between the first and 18th month of life (from 0.80 to 1.55 ml/min/kg in males; from 0.84 to 1.80 ml/min/kg in females). Similar changes occur in dogs and humans; the change is due to inadequate development of the hepatic microsomal enzyme system in the neonatal period. It was concluded that clear-cut sex differences in MRT and Cl occurred in cattle over eight months in age, the females being the more active metabolizers.
In a similar manner, Janus et al. (2001
) investigated the effects of short-term (4 days) starvation or water deprivation on the pharmacokinetics of caffeine (5 mg/kg, intravenously) in three groups of ten 24- to 25-day-old Holstein calves. An automated enzyme-multiplied immunoassay technique was used to determine plasma caffeine concentration just before the administration of caffeine and four days later at the end of the deprivation period. Results from the caffeine study indicated that four days of starvation or water deprivation was associated with significant increases in MRT and Total Plasma Clearance (Clt) of 20 to 30%. V
was slightly (not significantly) decreased. It was concluded that the results from this study were similar to the findings reported in sheep, horses, laboratory animals, and humans, and indicate that starvation and water deprivation lead to a general inhibition of the hepatic P450 enzyme system and may impair the elimination of drugs that undergo metabolism by these enzymes.
Pelissier-Alicot et al. (2002
) investigated the effects of caffeine on the daily rhythms of heart rate, body temperature, locomotor activity, and caffeine pharmacokinetics (PK) in 10-week-old male Wistar rats in relation to time-of-day. The study was divided into three 7-day phases: a control period, a treatment period, and a recovery period. During the treatment period, 25 mg/kg of caffeine was administered subcutaneously to groups of rats (four rats/group) at 8:00 AM in the morning, and to other groups at 8:00 PM in the evening. Blood for PK parameters was drawn at periodic intervals of 0.25 to 24 hr postinjection on the 7th day of treatment. Telemetry was used in similarly treated rats to obtain pharmacodynamics data. Morning administration of caffeine suppressed locomotor activity and modified the diastolic–systolic amplitudes of heart rate and body temperature; evening administration did not alter locomotion, but altered the blood pressure elevations, amplitudes, and acrophases of the three rhythms, indicating a chronopharmacologic effect. PK data revealed that the area-under-the-curve (AUC) was significantly lower in rats medicated in the evening, compared to medication in the morning, due to an increase in total plasma clearance and volume of distribution. However, there was no significant time of administration-dependent difference in Cmax
, or half-life.
The influence of hepatic regeneration after partial hepatectomy (removal of median and lateral lobes) on theophylline (Th) pharmacokinetics in groups of five adult male Wistar rats was studied by Maza et al. (2001
). At 12 and 24 hr and 3, 6, and 15 days after partial hepatectomy, Th was administered intravenously as a single dosage of 6 mg/kg, and plasma concentrations were determined at periodic intervals. Liver weights and clinical pathology parameters were also determined. Liver mass at the respective dates above were: 3.8, 5.0, 6.5, 7.1, and 9.4 g, compared to 12.1 g in nonhepatectomized rats. Liver function tests were increased significantly at 12 and 24 hr. Initial Th concentrations and volume at steady state varied during regeneration. The control elimination half-life of 4.30 ± 1.37 hr notably increased after hepatectomy (7.27 ± 1.38 hr), and then decreased with time to 5.17 ± 0.87 hr at 15 days. The increase in elimination half-life led to a decrease in mean residence time during the period of regeneration; however, the intrinsic clearance hardly varied.
Appropriate Use of Animal Studies for Assessing Human Risk
Although many metabolic and kinetic factors appear similar in rats and humans, only clinical studies in humans and intact animal pharmacokinetic studies in animals can be used to extrapolate risks from animal species to humans. There are few or no data regarding blood levels attained or the comparability of dosages administered. One of the most important considerations regarding comparability of blood levels is that humans consume caffeine over a period of time, rather than as a bolus dosage, and certainly not from an intraperitoneal injection. Humans consuming a 1 to 2 mg/kg dosage of caffeine attain a blood concentration of 1 to 2 µg/ml; a 3 to 5 mg/kg intake of caffeine results in a 5 µg/ml serum concentration. Thus, a 1 mg/kg intake produces a 1 µg/ml blood concentration over the range humans are likely to consume, fitting first-order kinetics for human metabolism of caffeine. The kinetics in rats is dose-dependent and zero order, indicating a saturable process, particularly at high dosages (Christian and Brent, 2001
) ( and ).
Many animal studies in the previous review (Christian and Brent, 2001
) and in this current review were conducted using bolus gavage dosages, rather than exposure over a period of time as the result of administration in the drinking water or diet. Such differences in the route of exposure often confound interpretation of data and results in inappropriate identification of the NOEL (no observable effect level). Most comparisons are made on the basis of mg/kg dosages, rather than attained blood levels, that are generally considered more useful in cross-species extrapolation, but which are rarely identified in human studies. For example, pregnant rats that were administered caffeine by gavage or via the drinking water for the first 11 days of pregnancy and then administered an 80 mg/kg dosage of radiolabeled caffeine on days 12 to 15 of gestation had blood serum concentrations of caffeine that were much greater after gavage dosage (60–63 µg/ml) than after drinking water exposure (0.10–5.74 µg/ml). However, the drinking water levels were more variable because of the remarkable variability in timing and consumption of drinking water. The half-life of an 80 mg/kg dosage of caffeine in pregnant rats in this study was approximately 1.7 to 2.6 hr (Christian and Brent, 2001
) ( and ). When two bolus gavage dosage of caffeine, 5 and 25 mg/kg, were administered to Wistar pregnant rats, apparent enzyme saturation resulted in nonlinear kinetics at the higher dosage only, resulting in an increased half-life and/or an increased distribution phase. However, mean peak plasma concentrations in nonpregnant and pregnant gestation day 20 rats and in the placenta, amniotic fluid, and fetal blood were linear at approximately equivalent times for both dosages. At 24 hr after the 25 mg/kg dosage, plasma concentrations of caffeine were 2 µmol/l (0.4 µg/ml) and 20 µmol/l (4 µg/ml) in nonpregnant and pregnant rats, respectively, and the half-life was significantly longer in pregnant (8.9 hr) than in nonpregnant (3.8 hr) rats at the 5 mg/kg dosage but increased at the 25 mg/kg/day dosage, indicating saturation (Christian and Brent, 2001
) ( and ). When given intravenously to pregnant sheep, as described by Tomimatsu et al. (2007
), maternal intravenous administration of 3.5 mg/kg of caffeine resulted in a maternal plasma caffeine concentration of 5 µg/ml and fetal caffeine concentrations in excess of 80% of maternal concentration. Other authors cited that the metabolism of caffeine differs between rats and humans, with the half-life much shorter in rats. Using a correction factor, Tanaka et al. (1983
) demonstrated that a dosage of 70 mg/kg/day ingested by pregnant rats is equivalent to a dosage of approximately 30 mg/kg/day for humans. Thus, Bodineau et al. (2003
) considered a 49 mg/kg/day dosage of caffeine in drinking water to pregnant rats to be in the moderate range for a human model although all other authors consider this a high exposure. Newborns exposed to caffeine in utero exhibited apnea postnatally.
In toto, these toxicokinetic experiments show that
- Serum and/or plasma concentrations of caffeine are much higher in rats after gavage treatment than after sipping treatment or continuous intravenous infusion;
- Pregnancy alters pharmacokinetics in both humans and rats, and
- The changes may be dose-dependent and species-specific.
Yet, pharmacokinetic studies with caffeine can serve a very useful purpose, especially when it is used as a biomarker for the estimation of xenobiotic biotransformation and possible hepatotoxicity. An example of such an investigation was conducted in adult mice (BALB/c mice) by Kolarovic et al. (1999
). The test article was enflurane, a fluorinated volatile anesthetic, administered by inhalation in either anesthetic or subanesthetic doses, with/without prior intraperitoneal injection of 1 g/kg ethanol. Two control groups were administered only ethanol or saline. Anesthetic exposure occurred for 6 hr/day for 5 days. On the 6th day, half the mice were injected intraperitoneally with 20 mg/kg caffeine and 8-hr urine samples were collected for caffeine metabolite assay; remaining mice were used to determine liver function and cytochrome P450 analysis. Liver function tests were all normal, but liver P450 levels were higher in the group treated with enflurane and ethanol, compared to other groups. Excretion of caffeine and its metabolites was different among the groups. Quantities of caffeine metabolites that are predominantly metabolized by CYP-4502E1 were higher in urine of enflurane-treated mice, while quantities of caffeine metabolites predominantly metabolized by CYP-4501A2 were significantly lower than in controls. Control values for the CYP-4501A2 enzymes were: 1,7-dimethyl uric acid (1,7-U) = 4.155 ± 1.956; 1,3,7-threemethyl uric acid (1,3,7-U) = 6.314 ± 2.992. It was concluded that use of caffeine as a biomarker is a highly sensitive test for estimating xenobiotic transformation and possible hepatotoxicity.
Caffeine Studies Relevant to Teratogenicity or SA (Pregnancy Loss)
Previous FDA (1980
) conclusions and those described by Christian and Brent (2001
) appear to provide sufficient precaution regarding consumption of caffeine, that is, that moderate consumption of caffeine (which was defined as ≤5–6 mg/kg/day) is unlikely to increase the risk of SA. These conclusions also appear to apply to the two additional human studies summarized below that were included in the present literature search conducted in 2008.
In a case–control study of 73 women with, and 141 women without SA, Fenster et al. (1998
) determined the activity of the three principal caffeine-metabolizing enzymes (P4501A2, xanthine oxidase, and N
-acetyltransferase) by measuring the levels of caffeine metabolites in urine. Caffeine was entered as a categorical variable in models with the following levels of caffeine consumption: no caffeine level; 1 to 150 mg/day (<2.5 mg/kg in a 60-kg woman); and >150 mg/day. Results established no association between caffeine consumption, caffeine metabolism, and risk of SA. However, due to small sample size, the study was not able to reliably estimate the risk for recurrent abortion in relation to caffeine consumption and the indices of enzyme activity.
Possible adverse effects of caffeine on pregnancy were also investigated by Klebanoff et al. (2002
). They tested 2,515 women to determine whether third-trimester maternal serum concentration of paraxanthine, caffeine's primary metabolite, is associated with the delivery of a small-for-gestational age infant (birth weight of <10th percentile for gender gestational age and ethnicity), and whether the magnitude of the association is affected by smoking. The subjects were selected from women who enrolled in the Collaborative Perinatal Project at 12 sites in the U.S.A. The mean serum paraxanthine concentration was greater in women who gave birth to small-for-gestational age infants (754 ng/ml) than to “normally” grown infants (653 ng/ml, p
= 0.02). However, the linear trend for increasing serum paraxanthine concentration to be associated with increasing risk of small-for-gestational age birth was confined to women who also smoked (p
= 0.03). There was no association between paraxanthine and fetal growth in nonsmokers (p
The Frog Embryo Teratogenesis Assay—Xenopus (FETAX) was used to test the 13 metabolites, including theophylline, paraxanthine, and a synthetic methylxanthine analogue (Fort et al., 1998
). Frog embryos were exposed to two concentrations of each test article, with or without a metabolic activation system. Assay results indicated that the fetotoxic potencies of each of the di- and monomethylxantine metabolites were similar to that of caffeine. None of the caffeine metabolites tested was found to be significantly more potent than caffeine itself in the FETAX assay.
Caffeine Interaction with Stress
A series of manuscripts were produced by researchers at the University of Seville, Spain and the University of Picardie Jules Verne, France (Bodineau et al., 2003
; Saadani-Makki et al., 2004
; Gaytan et al., 2006
) regarding the potential effects of caffeine and other xyanthines as the result of their binding with adenosine receptors and their potential effect on respiration. Again, these studies were conducted because caffeine is used therapeutically to normalize breathing in apnea-affected infants. The authors stated that premature infants may be exposed to relatively high serum concentration of caffeine (10–15 µg/ml) for up to 8 weeks of treatment. They referenced Shi et al. (1993
) who demonstrated that chronic caffeine exposure alters the density of adenosine, adrenergic, cholinergic, GABA, and serotonin receptors and calcium channels in the mouse brain, resulting in a reduction in the fetal cerebral weight. They also indicate that sustained maternal caffeine intake induces harmful physiologic effects on human newborns, including respiratory perturbations, citing a case report (Khanna and Somani, 1984
) of a woman reported to have consumed 24 cups of coffee per day during pregnancy, with a newborn who experienced apnea episodes attributed to methylxanthine withdrawal.
The first study by Bodineau et al. (2003
) was conducted using the drinking water route (calculated consumed caffeine dosage = 49 ± 4 mg/kg/day). A subsequent study by the same group (Saadani-Makki et al., 2004
) used tissues from the generated pups and evaluated brainstem–spinal cord preparations isolated from these newborn rats. In both studies, the authors noted an increase in pup weight, without any consideration for the mean number of pups per litter. Both these observations should be considered unrelated to caffeine [the increase in newborn weight (7.7 g) in the caffeine exposed group versus the control (6.7 g) was most probably the result of the fewer pups in the caffeine group (10.9 pups) versus the control (13.8 pups), a finding reflecting the relatively few litters evaluated (eight per group) and the normal variability in litter sizes]. No historical data were provided.
In the Bodineau et al. (2003
) study, the consequences of in utero caffeine exposure on respiratory output in normoxic and hypoxic conditions and related changes of Fos (binding protein involved in transcription regulation) expression were evaluated. The study was conducted using brainstem–spinal cord preparations isolated from newborn rats. Sprague–Dawley rats (control and caffeine groups = 8/group) were given water or 0.02% caffeine in water, with intake evaluated daily, presumably from conception until parturition, because the caffeine was removed after parturition. The experiments were conducted on brainstem–spinal cord preparations isolated from 37 control and 35 caffeine group rats. The authors claimed to know the exact dosage consumed (50.4 ml/day control, 62.3 ml/day—caffeine) with the consumption of 49 ± 4 mg/kg/day, estimated according to drinking fluid intake. The body weight was increased and litter size of the newborn caffeine group rats was reduced, compared with the control group. However, based on the standard deviation of the caffeine group, it is probable that one litter was affected (data were not provided; and the authors did not identify statistical significance). A later study (Saadani-Makki et al., 2004
), using tissues from the same animals, further evaluated involvement of the adenosinergic A1 systems in the occurrence of respiratory changes in newborns after in utero caffeine exposure and the importance of the rostral pons in adenosinergic A1 modulation in respiratory control. As before, exposure was during pregnancy, via maternal drinking water, and caffeine fluid intake was estimated at 49.8 mg/kg/day, based on drinking fluid intake, a toxic level. The authors concluded that their work brought evidence of the involvement of the adenosinergic A1 systems in the occurrence of apnea in newborn infants after in utero caffeine exposure.
Further studies by this group (Gaytan et al., 2006
) evaluated postnatal exposure to caffeine on the pattern of adenosine A1 receptor distribution in respiration-related nuclei of the rat brainstem. They evaluated the ontogeny of the adenosine A1 receptor system in the brainstem of the newborn rat after postnatal treatment with caffeine. This study identified that the previously reported results, with the main difference between control and caffeine administered rats being the transient increase (on postnatal day 6 only) in the parabrachial and Kõlliker–Fuse nuclei, which are classically associated with the adenosine A1 receptor system. The authors concluded that the role of caffeine in decreasing the incidence of neonatal respiratory disturbances may be due to earlier than normal development of the adenosinergic system in the brain.
There was another group of publications originating in Spain regarding the potential interactions of caffeine and stress during pregnancy in mice (Colomina et al., 2001
; Albina et al., 2002
). In the manuscript by Colomina et al. (2001
), a single oral dosage of caffeine or aspirin on p.c.d 9 was given to mice orally exposed to toxic levels of caffeine (30 mg/kg/day), aspirin (250 mg/kg), or a combination of caffeine and aspirin (30 and 250 mg/kg, respectively). Three additional groups were given the same doses and restrained for 14 hr. The pregnant mice were restrained 2 hr/day on p.c.ds 0 to 18 by placing them in methacrylate cylindrical holders and keeping them in a prone position with the paws immobilized with elastic adhesive tape, a procedure the authors previously reported to produce stress in pregnant mice (Colomina et al., 1995
; Scialli et al., 1995
; Colomina et al., 1999
). Other mice were given toxic dosages of caffeine by gavage at 30, 60, and 120 mg/kg/day on GDs 0 to 18, and another group was administered the same dosages of caffeine immediately followed by restraint stress for 2 hr/day on the same days (Colomina et al., 1999
). No caffeine levels were recorded. Although the authors do not identify maternal toxicity, it is noteworthy that the weekly intervals measured for body weights are inappropriate (drug treatments and restraint occurred on one day; the intervals are evaluated for three or four days). Maternal toxicity was evident, with reductions or frank weight losses in body weight and feed consumption measurements. Regarding caffeine, these effects were most severe for the three groups of interest (restraint, 30 mg/kg caffeine and combined 30 mg/kg of caffeine and 14 hr of restraint), on p.c.ds. 9 to 11. Of these three groups, the effects were most severe for the combined caffeine and stress group. The 30 mg/kg plus restraint group also had an increase in postimplantation loss, including dead fetuses and late resorptions. An increase in early resorptions was seen in the restraint alone group, but the group with both restraint and 30 mg/kg of caffeine were increased compared with the restraint alone group. As would be expected, there was an increase in reduced ossification in the restraint group alone, the 30 mg/kg caffeine alone, and the combined caffeine and stress group. There was no increase in malformations in any group. The authors considered there to be some clinical relevance for the data because real life involves multiple simultaneous exposure to many chemicals. However, the duration of oral exposure to aspirin and caffeine on gestational day 9 in this study is not analogous to the type of stress experienced by pregnant women who drink coffee and take aspirin. Interspecies differences and pharmacokinetics and bioavailability are both important consideration.
Albina et al. (2002
) reported a study by Nehling and Debry (1994
) in which daily consumption of caffeine ranged from 203 to 283 mg, or 2.7 to 4.0 mg/kg/day of caffeine in adults (equivalent to 3.38–4.72 mg/kg for a 60 kg person). Albina et al. (2002
) also refer to the FDA 1980
recommendation that pregnant women limit caffeine consumption to less than 400 mg/day (6.7 mg/kg/day for a 60 kg human), based on animal studies (FDA, 1980
). These authors report that 30 mg/kg/day of caffeine administered with maternal stress is an effect level (they did not report that stress alone was an effect level). Nonetheless, the authors recommended that women under notable stress during pregnancy should reduce caffeine ingestion to reasonable levels; for example, a dosage of 10 mg/kg/day. For 60-kg women, 10 mg/kg/day would be a daily ingestion of 600 mg, or four cups of strong coffee or eight cups of weak coffee.
Interaction of Caffeine as a Pharmaceutical
A series of studies in rats was conducted by Burdan and his colleagues at the Experimental Teratology Unit of the Human Anatomy Department of the Medical University School in Lublin, Poland. The initial objective was to evaluate the effects of caffeine on skeletal development, when administered by gavage during gestation (Burdan et al., 2000
). The later studies were designed to evaluate the effects of over-the-counter preparations of various mixtures of propyphenazone, caffeine, and paracetamol, with the purpose of determining liver toxicity (Burdan et al., 2001
) and the prenatal risk of COX inhibitors administered with or without caffeine (Burdan, 2002
). The studies were conducted in general conformance with evaluations performed for testing pharmaceuticals, but used fewer rats than are usually utilized in studies designed for regulatory use (generally 15 per group, rather than the recommended 16–20 litters), an abbreviated treatment period p.c.ds. 8 to 14, rather than the current usual interval, gestation days 7 to 17. As a result, the exposure period differs by one day from many studies published for regulatory use. Nevertheless, the manuscripts are well documented and easily interpreted. All the findings regarding caffeine's maternally and developmentally toxic dosages do not indicate new concerns, even in combination with the interacting medications.
Burdan et al. (2000
) did not observe adverse maternal or developmental effects at caffeine dosages up to 70 mg/kg administered on p.c.ds. 8 to 14, which is unusual. The Burdan et al. (2001
) study showed that caffeine is toxic to the liver only at dosages greater than those tested in this study (the highest dosage of caffeine tested was 70 mg/kg/day), and when given for a prolonged period. The dosages tested in this study were mixtures prepared in 5:3:1 ratio (acetaminophen, isopropylantipryine, and caffeine), with the caffeine dosages at 0.7, 7, and 70 mg/kg/day. Although the authors concluded that the administration of the mixture to nonpregnant rats at the maximum dosage tested in this study only slightly impaired liver function, hepatotoxic effects were observed in pregnant female rats at the high dosage. Thus, they also concluded that the pregnant rat's liver was more vulnerable than the nonpregnant rat's to the tested materials, although they cautioned that the studies were difficult to extrapolate to human exposure.
The next series of studies of combined drugs in over-the-counter products evaluated acetaminophen, isopropylantipyrine, and caffeine (Burdan, 2002
). There were 29 control rats and 15 to 19 per group in those administered the caffeine mixture. Caffeine was given by gavage at 0.7, 7.0, or 70 mg/kg, in combination with the other drugs (acetaminophen:isopropylantipyrine:caffeine 5:3:1 ratio [A:I:C]). The authors concluded that this mixture of acetaminophen, isopropylantipyrine, and caffeine administered in a constant proportion of 5:3:1 for the entire second week of pregnancy was not teratogenic in rats but was maternally toxic at the mid and high dosages (35:21.4:7 and 350:214:70 mg/kg, respectively), and was embryotoxic only at the high dosage (350:214:70 mg/kg, respectively).
) then administered dosages of 3.5:0.7, 35.0:7.0, and 350:70 mg/kg/day of paracetamol:caffeine, respectively, on p.c.ds. 8 to 14. All dosages were maternally toxic, producing reduced maternal weight gain and liver weight. The mid and high dosages also reduced kidney weights, observations that were attributable to paracetamol. At the maternally toxic mid and high dosages, reduced fetal body weight/growth and placental weight occurred, previously described reversible effects of gavage dosages of caffeine, but there was no increase in fetal malformations.
Finally, Burdan (2004
) administered maternal dosages of 2.1:0.7, 21:7, or 210:70 mg/kg prophyphenazone:caffeine on p.c.ds. 8 to 14. The only evidence of maternal toxicity was decreased liver weight at the high dosage. Fetal body weight was reduced in groups given the middle (21:7 mg/kg prophyphenazone:caffeine) and high (210:70 mg/kg prophyphenazone:caffeine) dosages of the propyphenazone:caffeine mixtures and the middle dosage of the propyphenazone:paracetamol mixture. The effects on fetal body weight were not dose-dependent, possibly because of the increase in resorption that also occurred at the high dosage. These results are similar to the previously described parallel studies in which all three compounds were given separately or in a mixture. Dose-dependent liver injury was seen in dams given propyphenazone and caffeine and the mixture with all three ingredients, with a hepatotoxic effect and decrease in maternal body weight in the middle and high dosage groups. The authors concluded that co-administration of propyphenazone and caffeine or propyphenazone and paracetamol caused growth retardation but no teratogenic effects and that the results supported the prenatal safety of low dosages of caffeine.
Caffeine Studies Regarding Adenosine Receptor Interaction and Adenosine Effects
As noted in some of the studies previously discussed, caffeine interacts with the adenosine receptor, and it is the most widely known adenosine receptor antagonist. The biochemical mechanism underlying the effects of caffeine is the blockade of adenosine receptors, which is an antagonist for adenosine modulation. Although adenosine receptor interaction wth caffeine may not result in teratogenicity, caffeine may affect neuronal growth and neuron interconnections during gestation and the neonatal period. It would be important to determine the NOAEL for deleterious effects on neuronal growth and neuron synapse formation.
Caffeine modulation of adenosine receptor and ontogeny was tested in the following studies. Snyder (1984
) provided an extensive review of adenosine as a potential mediator of the behavioral effects of xanthines, approximately 20 years after it was identified that phosphodiesterase was an enzyme that degraded cyclic AMP (Sutherland and Rall, 1958
; Butcher and Sutherland, 1962
; Salmi et al., 2007). According to Iglesias et al. (2006
), adenosine, a nucleoside, is widely distributed in the peripheral and central nervous systems and acts through G-protein coupled receptors. Four types of receptors have been identified: A1, A2A, A2B, and A3. A1 and A3 receptors inhibit adenylyl cyclase activity through Gi protein. A2A and A2B receptors act by stimulating adenylyl cyclase activity through Gs protein. A1 and A2A receptors have a greater affinity with adenosine and are blockaded by caffeine. Adenosine, working through the A1 receptors, inhibits glutamate release, thus acting as a neuromodulator and neuroprotector. Snyder (1984
) also noted that phosphodiesterase was inhibited by the xanthines, including caffeine and theophylline, and that via this mechanism, xanthines could elevate cyclic AMP levels. However, to substantially inhibit phosphodiesterase, millimolar concentrations of caffeine were required, approximately 100 times the levels of caffeine found in the human brain after ingestion of typical dosages in humans. In addition, it was noted that some inhibitors of phosphodiesterase were 100 to 1,000 times more potent than caffeine but without behavioral effects.
Adenosine has many effects, including dilation of blood vessels, especially in the coronary and cerebral circulation, inhibition of platelet aggregation, and inhibition of hormone-induced lipolysis. It also has a variety of actions on central neurons, usually inhibiting spontaneous neuronal firing (Phillis and Wu, 1981
; Stone, 1981
). Adenosine inhibition of the release of excitatory neurotransmitters is the predominant presynaptic activity, although postsynaptic effects are also present. Many studies were conducted testing the hypothesis that in utero exposure altered adenosine receptors and their activities, including postnatal functional activity in the brain and heart. All the studies appear to have been performed at dosages that either were toxic, were reversible in effect, or not sufficiently well documented for use in human risk assessment.
The first biochemical analysis of adenosine receptor activity was by Sattin and Rall (1970
) who demonstrated that adenosine can increase the accumulation of cyclic AMP in brain slices without conversion of adenosine to cyclic AMP, an action on extracellular receptors. The effects of adenosine on the enzyme adenylate cyclase, which synthesizes cyclic AMP, revealed two distinct subtypes of adenosine receptors, designated A1 and A2 (van Calker et al., 1979
; Burnstock and Brown, 1981
; Londos et al., 1981
). Depending upon the system, adenosine increases or decreases adenylate cyclase activity, with the enhancing actions occurring at micromolar concentrations via A2 receptors. Nanomolar concentrations of adenosine cause the A1 receptors to inhibit adenylate cyclase activity. Marked sterospecific effects of phenylisopropyladenosine (PIA) occurs at the A1 receptors. L-PIA is remarkably more potent than D-PIA, although the two isomers are relatively similar in effect at the A2 receptors. Most xanthines have similar potencies blocking both A1 and A2 receptors.
Direct binding studies have demonstrated that in all species studied, adenosine receptors labeled with [3H]DPX, a xanthine derivative, binding showed that nanomolar potency was present for adenosine derivatives and sterospecificity for PIA isomers. However, binding studies identified heterogeneity of adenosine receptors beyond the A1 and A2 distinction. Another xanthine derivative (DPX) was about 250 times more potent in competing for [3H]CHA sites in calf than in guinea pig and human brain. As summarized by von Borstel and Wurtman (1984
), considerable evidence has been accumulated that competitive antagonism at cell surface adenosine receptors may be the most important molecular action for methylxanthines, including caffeine. Administration to animals can produce sedation, bradycardia, hypotension, hypothermia, and attenuation of the response of the heart, vascular, and adipose tissue to sympathetic stimulation and are generally opposite to those produced by caffeine or theophylline alone. Methylxanthines competitively antagonize these and other adenosine actions at concentrations similar to those found in plasma after consumption of one to three cups of coffee (5–30 µM) (Rall, 1980
A series of new manuscripts identified in this review describe studies designed to evaluate the effect of caffeine on adenosine receptor ontogeny. One group of investigators (Adén et al., 2000
) identified that administration of caffeine at dosages resembling those consumed by humans does not significantly influence the development of receptors known or believed to be affected by caffeine. The results, described below, in contrast to other publications, indicate that caffeine can modify adenosine receptors and/or behavior. However, it is unclear what dosages were used or what postnatal blood levels of caffeine were attained. Adén et al. (2000
) reported that maternal caffeine intake has minor effects on the adenosine receptor ontogeny in the rat brain. Caffeine was provided in the drinking water given to pregnant rats, beginning on p.c.d. 2 and continuing throughout gestation and postnatal life of the offspring. Although the authors noted that only a low dosage of caffeine was administered, estimated to be up to 3 cups of coffee/day, or what a woman might drink during pregnancy, it must be noted that mg/kg/day consumed dosages vary throughout gestation and lactation. This is further confounded by the pup's consumption of the maternal drinking water, which contained caffeine. They reported that low-dosage caffeine-exposure during gestation and postnatal life had minor effects on the development of adenosine A1 and A21 receptors and GABAA receptors in the rat brain.
Other studies were often designed to evaluate whether caffeine affected excitotoxic brain lesions in mice, because it is often given to human pre-term newborns. Bahi et al. (2001
) examined the effects of caffeine on neonatal excitotoxic lesions of the periventricular white matter. This study was designed to mimic caffeine exposure of human preterm infants in neonatal intensive care units. Most of this study is inappropriate for inclusion in this review because it addresses postnatal evaluations, rather than in utero exposure. It has been included because it had two sets of experiments, one performed postnatally and the other with in utero exposure, unfortunately by the intraperitoneal route (5 mg/kg caffeine citrate administered IP to 3 pregnant dams on p.c.ds. 8–18 and another group injected IP with 12.5 mg/kg caffeine on p.c.ds. 8–11). Although no mechanism was shown, it appeared that caffeine had a neuroprotective effect in mice.
An interaction study in knock-out mice was performed by Björklund et al. (2007
) to investigate whether the response of the adenosine receptor system to a low perinatal exposure to methylmercury (MeHg) would be altered by caffeine treatment or eliminated by genetic modification (A1R and A2AR knock-out mice). Pregnant mice were administered 1 µM MeHg and/or 0.3 g/l caffeine (>30 mg/kg) in the drinking water. The consequences of MeHg toxicity during gestation and lactation were reduced by adenosine A1 and A2a receptor inactivation, either by genetic deletion or treatment with their antagonist, caffeine. This work also showed a protective effect of a high caffeine dosage of (>30 mg/kg/day).
In a 2008 study, da Silva et al. evaluated maternal caffeine intake to determine whether it affected acetylcholinesterase in the hippocampus of neonatal rats. The control group was given tap water, and the caffeine group given 1.0 g/l caffeine diluted in tap water. Experiments were performed using 30 male and 30 female pups at 7, 14, and 21 days of age. Caffeine did not change the age-dependent increase of acetylcholinesterase activity or the age-dependent decrease of acetylcholinesterase expression. However, it resulted in a 42% increase in acetylcholinesterase activity, without changing the level of acetylcholinesterase mRNA transcripts in 21-day-old rats. These results further demonstrate the ability of maternal caffeine intake to interfere with cholinergic neurotransmission during brain development.
A series of studies conducted by investigators in Spain considered the effects of down regulation of Adenosine A1 receptors and other receptors in the brain and heart that are affected by caffeine (León et al., 2002
; Iglesias et al., 2006
). They reported caffeine intake as 83.2 mg/kg/day (administered at 1 g/l in the drinking water from p.c.ds. 2 throughout pregnancy [sperm = gestation day 1]). The reported estimated dosage appears to be correct, because a 250 g rat would consume at least 20 ml/day of drinking water, although this value is somewhat low for a pregnant rat. These investigators considered this dosage equivalent to approximately 80 to 180 mg caffeine in a cup of coffee, or consumption of one cup of coffee by a pregnant woman. This calculation appears inappropriate because 180 mg consumed by a 60-kg human would be equivalent to only 3 mg/kg/day, much lower than the 83.2 mg/kg/day dosage consumed by the rats.
In the León et al. (2002
) publication, it was reported that caffeine consumption during gestation caused down-regulation of adenosine A1 receptors in both the maternal and fetal brain. The later publications noted that it also inhibited A1 receptor function in the maternal rat brain and down regulation of metabotropic glutamate receptors in the brain from both mothers and fetuses (Leon et al., 2005a
). The results of this study, evaluating isolated rat heart membranes, immunodetection of mGluR1, indicate down-regulation of different components of the mGlur I/PLC pathway in the maternal and fetal heart, and loss of receptor responsiveness in fetuses that can alter the physiological function of the heart, especially in fetal tissue mGluRs.
Iglesias et al. (2006
) demonstrated that chronic intake of caffeine during gestation in rats down regulates metabotropic glutamate receptors in maternal and fetal rat heart. While most of the studies involve the interaction of caffeine with adenosine receptors (Sutherland and Rall, 1958
; Butcher and Sutherland, 1962
; Snyder, 1984
; Iglesia et al., 2006
) caffeine also interacts with adrenergic, cholinergic, GABA, and serotonin receptors as well as calcium channels (Shi et al., 1993
Keller et al. (2007
) provided an excellent review of cardiovascular development in which maternal exposure to hypoxic and bioactive chemicals, for example, caffeine, can rapidly impact embryonic/fetal cardiovascular function, growth, and outcome. No specific description of caffeine exposure in animals or humans was provided.
A study by Asadifar et al. (2005
), while not relevant to toxicity produced as the result of in utero exposure of pregnant rats to caffeine, addresses the interaction of combined effects of caffeine and malnutrition on Cu content in the neonatal rat heart.The results of this study, in which neonates were administered a normal diet with 20% protein, 20% protein supplemented with caffeine (4 mg/100 g BW) or 6% protein diet (malnourished) or 6% protein supplemented with caffeine (4 mg/100 g BW) from birth to postnatal day 10 were surprising and not what was expected. The caffeine level was considered comparable to consumption of a heavy coffee drinker, defined as 4 cups of coffee containing an average of 100 mg of caffeine and an average body weight of 50 kg (400 mg/50 kg = 8 mg/kg). The results show that malnutrition did not impair mitochondria, and that although it was expected that caffeine exposure would aggravate their Cu status, the results were the opposite of the hypothesis. Caffeine exposure affected Cu status more in the normally nourished animals than in the malnourished animals, an apparent protective effect.
Momoi et al. (2008
) further evaluated maternal and embryonic cardiovascular function in CD-1 mice administered 10 mg/kg/day caffeine subcutaneously on p.c.ds. 9.5 to 18.5 of a 21-day pregnancy period (this information appears in error, because mice have an 18-day pregnancy). Blood levels were not reported, so it is not possible to extrapolate to human exposure, although the authors considered the exposure to be equivalent to modest daily maternal exposure. (It should be noted that the caffeine was administered by injection rather than by oral administration in the diet, so it is unlikely that this exposure was comaparable to human caffeine exposures.) No maternal toxicity or increase in embryo resorption was observed. At p.c.d. 18.5, crown-rump length, forelimb length, and wet body weight of caffeine-treated embryos were smaller than the control embryos. The main findings of the study were reported as: (1) modest daily maternal caffeine exposure altered regional developing embryonic arterial blood flow and induced intrauterine growth retardation without impacting maternal CV function or weight gain; (2) caffeine at peak maternal serum concentration transiently reduced embryonic carotid arterial flow to a greater extent than dorsal (and descending) aortic or umbilical arterial flow; (3) maternal adenosine A2A receptor blockade reproduced the embryonic hemodynamic effects of maternal caffeine exposure; and (4) adenosine A2A receptor gene expression in the uterus and developing embryo were down regulated by maternal caffeine exposure. The authors considered the 10 mg/kg dosage of caffeine to be a modest maternal caffeine dosage. They also stated that maternal caffeine effects in a mouse model may not reflect human effects, and concluded that modest daily maternal caffeine exposure may have a negative effect on embryonic CV function and overall embryonic growth, possibly mediated by adenosine A2A receptor blockade.
Another study in near-term fetal sheep (Tomimatsu et al., 2007
) was performed to test the hypothesis that maternal caffeine administration does not significantly alter fetal cerebral oxygenation. The authors considered the dosage comparable to one that may be consumed by pregnant women in daily life. The pregnant ewes and their fetuses were instrumented at post conception day 125 ± 3 (term ~ 145 days). A total of 800 mg of caffeine citrate (400 mg of caffeine, reported as approximately 8 mg/kg, that is, equivalent to 2–3 cups of coffee) into the maternal inferior vena cava over 30 min. Fetal arterial and sagittal sinus blood samples and maternal arterial samples were collected every 10 to 15 min and analyzed for blood gases, hemoglobin concentration, oxyhemoglobin saturation, and calculated O2
content. Maternal parameters were unaffected. Fetal arterial blood gas values at 5, 30, and 40 min after the 30-min maternal infusion of caffeine were also not significantly affected. However, sagittal sinus O2
content and oxyhemoglobin saturation were significantly decreased in fetuses, although neither fetal heart rate nor mean arterial blood pressure were significantly changed. After 30 min of maternal caffeine infusion, fetal LD-CBF decreased slightly (− 7%). Fetal cortical PO2 decreased, and arterial to sagittal sinus O2
, content difference, cerebral fractional O2
extraction, and CMRO each increased 20 to 30% above baseline. Authors concluded that the results of their study showed findings that would suggest a small compromise in cerebral oxygenation occurred without affecting overall fetal systemic oxygenation. Further studies are needed to determine whether there are any related clinical findings.
Potential Model for the Production of Cataracts
Two publications (Evereklioglu et al., 2003
) reported results from the same set of rats. The Evereklioglu et al. (2003
) study was designed to identify whether histopathology could reveal changes in the neonatal rat cornea resulting from caffeine exposure during pregnancy. The Evereklioglu et al. (2004
) study focuses on the examination of the crystalline lenses in neonatal rats. Unfortunately, the study methodology was not well reported, and some tabular errors are evident, which preclude appropriate independent interpretation of the results.
Wistar pregnant rats and the i.p. route were used to treat a control and three dosage groups. As the result of the use of a route that is inappropriate for extrapolation to human exposure (i.p. dosages of 25, 50, and 100 mg/kg/day were administered between p.c.ds. 9–21), exposure relevant to human exposure comparisons cannot be made. A fifth group was given caffeine via gavage at a toxic dosage of 50 mg/kg/day. Dams delivered normally (generally on p.c.ds 20–21). Half of the newborn rats per litter were decapitated at postnatal day 1, and the eyes were examined. The remaining litters were raised with their biological mothers and sacrificed and decapitated at postnatal day 30 for eye evaluation. Pups were evaluated on postnatal days 1 or 30, and the eyes enucleated for corneal histopathology. Although the investigators refer to “pup” and “groups” and statistical analysis of these, it is somewhat unclear how this occurred because it appears that only one randomly selected eye (right eye) was evaluated. Thus, it appears that each litter and dosage group is represented by only one pup and one eye at each time interval.
No maternal toxicity was reported; however, 7 pups were reported as “miscarried” by 2 dams in 100 mg/kg/day caffeine Group 4 (high dosage), because rats do not generally abort but resorb their dead conceptuses. These “late fetal deaths” were probably either a sequela of IP injection and/or apparent premature delivery associated with incorrect identification of the mating date. Pup body weights were slightly decreased in all groups in a dose-dependent pattern. It is unclear whether the number of litters evaluated included the aborted litter at birth, or whether these litters were included with those with pups evaluated on postnatal day 30. in the Everekiloglu et al. (2003
) publication appears to incorrectly report the number of pups per litter as the mean number of pups per litter at birth. The authors concluded dosages of 50 mg/kg/day and higher affected development of the cornea, particularly postnatal at 100 mg/kg/day. Interestingly, macroscopic changes were not observed in any corneas on postnatal day 30.
In the later publication regarding effects in the same rats (Everekilioglu et al., 2004
), the ultimate objective was to establish a model for the study of cataract development, specifically, to investigate histologically the influence of maternal caffeine exposure during pregnancy on the development of the crystalline lenses in neonatal rats. In the control and 25 mg/kg/day dosage groups, both slit-lamp biomicroscopic and histopathologic examination of the crystalline lenses revealed normal findings. Histological examination of the 50 and 100 mg/kg/day IP groups and the 50 mg/kg/day PO group had findings suggesting cataractogenesis, including eosinophilic degeneration, lens fiber cell swelling and liquefaction, central lens fibers with retained nuclei, and prominent epithelial cells lining the posterior lens capsule behind the equator. Some lenses in the intraperitoneal 100 mg/kg/day group had immature cataract on slit-lamp biomicroscopic examination at postnatal day 30. The authors concluded that excessive maternal caffeine exposure during pregnancy had cataractogenic effects. As previously reported, no macroscopic ocular abnormalities were observed in control or experimental groups at birth, and the i.p. administration of high doses of caffeine prevents the ability to perform a valid risk assessment in humans.