Teratogenicity of paraxanthine (1,7-dimethylxanthine) in C57BL/6J mice

The teratogenicity of caffeine, as well as two of its three dimethylated metabolites (theobromine and theophylline), has been established in animal studies. The third metabolite, paraxanthine, has not been reported as being tested for teratogenicity even though it is actually the major demethylated metabolite of caffeine metabolism in man. Pregnant C57BL/6J mice were treated i.p. with 175 or 300 mg/kg/day paraxanthine (1,7-dimethylxanthine) dissolved in deionized water at 4 p.m. on day 11 and 9 a.m. on day 12 of gestation. All dams were sacrificed on day 18, and fetuses were fixed for Wilson's razor blade sectioning or double-staining skeletal examination. A dose-related increase in total malformations, primarily cleft palate and limb malformations, was found. The pattern of malformations was similar to that reported for caffeine, theobromine, and theophylline, i.e., an asymmetric response with the left forelimb most often affected. A 21% resorption and a 46% malformation rate was observed at 300 mg/kg/day of paraxanthine, indicating that paraxanthine was slightly less toxic to the embryo than caffeine. Therefore, the parent compound, caffeine, as well as all three of its dimethylated metabolites--paraxanthine, theophylline, and theobromine--are teratogenic.     

Biosynthesis of caffeine by tea-leaf extracts. Enzymic formation of theobromine from 7-methylxanthine and of caffeine from theobromine.

1. Extracts prepared from tea leaves with Polyclar AT (insoluble polyvinylpyrrolidine) contained two methyltransferase activities catalysing the transfer of methyl groups from S-adenosylmethionine to 7-methylxanthine, producing theobromine, and to theobromine, producing caffeine. 2. The methyltransferases exhibited the same pH optimum (8.4) and a similar pattern of effects by metal ions, thiol inhibitors and metal-chelating reagents, both for theobromine and caffeine synthesis. Mg2+, Mn2+ and Ca2+ slightly stimulated enzyme activity but they were not essential. Paraxanthine was shown to be most active among methylxanthines, as the methyl acceptor. However, the formation of paraxanthine from 1-methylxanthine was very low and that from 7-methylxanthine was nil, suggesting that the synthesis of caffeine from paraxanthine is of little importance in intact plants. Xanthine, xanthosine, XMP and hypoxanthine were all inactive as methyl acceptors, whereas [2(-14)C]xanthine and [8(-14)C]hypoxanthine were catabolized to allantoin and urea by tea-leaf extracts. The apparent Km values are as follows: 7-methylxanthine, 1.0 times 10(-14)M; theobromine, 1.0 times 10(-3)M; paraxanthine, 0.2 times 10(-3)M; S-adenosylmethionine, 0.25 times 10(-4)M (with each of the three substrates). 3. The results suggest that the pathway for caffeine biosynthesis is as follows: 7-methylxanthine leads to theobromine leads to caffeine. In contrast, it is suggested that theophylline is synthesized from 1-methylxanthine. The methyl groups of the purine ring of caffeine are all derived directly from the methyl group of S-adenosylmethionine. Little is known about the pathways leading to the formation of 7-methylxanthine. 4. A good correlation between caffeine synthesis and shoot formation or growth of tea seedlings was shown, suggesting that the methylating systems in caffeine synthesis are closely associated with purine nucleotide and nucleic acid metabolism in tea plants.     

Degradation of caffeine and related methylxanthines bySerratia marcescens isolated from soil under coffee cultivation

A strain of Serratia marcescens showing the ability to degrade caffeine and other methylxanthines was isolated from soil under coffee cultivation. Growth was observed only with xanthines methylated at the 7 position (caffeine, 1,3,7-dimethylxanthine; paraxanthine, 1,7-dimethylxanthine; theobromine, 3,7-dimethylxanthine and 7-methylxanthine). Paraxanthine and theobromine were released in liquid medium when caffeine was used as the sole source of carbon and nitrogen. When paraxanthine or theobromine were used, 3-methylxanthine, 7-methylxanthine, and xanthine were detected in the liquid medium. Serratia marcescens did not grow with theophylline (1,3-dimethylxanthine), 1-methylxanthine, and 3-methylxanthine, and poor growth was observed with xanthine. Methyluric acid formation from methylxanthines was tested in cell-free extracts by measuring dehydrogenase reduction of tetrazolium salt in native-polyacrylamide gel electrophoresis gel. Activity was observed for all methylxanthines, even those with which no bacterial growth was observed. Our results suggest that in this strain of S. marcescens caffeine is degraded to theobromine (3,7-dimethylxanthine) and/or paraxanthine (1,7-dimethylxanthine), and subsequently to 7-methylxanthine and xanthine. Methyluric acid formation could not be confirmed. Correspondence to: Paulo Mazzafera.     

Discriminative stimulus properties of methylxanthines and their metabolites in rats

Rats were trained to discriminate methylxanthines from saline under a two-lever concurrent variable ratio schedule of reinforcement. One group was trained to discriminate between saline and 32 mg/kg caffeine. A second group was trained to discriminate between 56 mg/kg theophylline and saline. Rats reliably discriminated between saline and the training methylxanthine, displaying graded generalization curves across training-drug doses. Caffeine-trained rats demonstrated caffeine-appropriate responding when tested with theophylline, paraxanthine, and 3-methylxanthine. Theobromine failed to generalize to the caffeine cue at test doses up to 75 g/kg. In contrast to the caffeine group, rats trained to discriminate theophylline from saline were less sensitive (higher ED50) to the effects of caffeine and paraxanthine test doses. Only partial generalization to the theophylline cue occured at paraxanthine doses up to 100 mg/kg. Based upon these data, it is suggested that the underlying substrate(s) for the caffeine cue is in some respects different from the substrate(s) for the theophylline cue.     

N-methyltransferases and 7-methyl-N9-nucleoside hydrolase activity in Coffea arabica and the biosynthesis of caffeine

The incorporation of radioactivity from L-[¹⁴CH₃]-methionine into caffeine by coffee fruits was enhanced by additions of theobromine and paraxanthine but was reduced by additions of theophylline and caffeine. Cell-free extracts prepared from seedlings, partially ripe and unripe coffee fruits showed that only the unripe green fruits contained significant methyltransferase and 7-methyl-N⁹-nucleoside hydrolase activity. The cell-free extracts catalysed the transfer of methyl groups fromS-adenosyl-L-[¹⁴CH₃]-methionine to 7-methylxanthine, and 7-methylxanthosine, producing theobromine and to theobromine producing caffeine. The two enzymic methylations exhibited a sharp pH max at 8.5 and a similar pattern of effects with metal chelators, thiol reagents and Mg²⁺ ions, which were slightly stimulating though not essential to enzyme activity. Paraxanthine (1,7-dimethylxanthine) was sh own to be the most active among methylxanthines as methyl acceptors; however its formation from 1-methylxanthine and 7-methylxanthine was not detectable, and biosynthesis from paraxanthine in the intact plant would therefore appear not to occur. The apparent K_m values are as follows: 7-methylxanthine 0.2 mM, theobromine 0.2 mM, paraxanthine 0.07 mM and S-adenosyl-L-methionine with each substrate 0.01 mM. The results suggest the pathway for caffeine biosynthesis in Coffea arabica is: 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine.     

Novel, highly specific N-demethylases enable bacteria to live on caffeine and related purine alkaloids

The molecular basis for the ability of bacteria to live on caffeine as a sole carbon and nitrogen source is unknown. Pseudomonas putida CBB5, which grows on several purine alkaloids, metabolizes caffeine and related methylxanthines via sequential N-demethylation to xanthine. Metabolism of caffeine by CBB5 was previously attributed to one broad-specificity methylxanthine N-demethylase composed of two subunits, NdmA and NdmB. Here, we report that NdmA and NdmB are actually two independent Rieske nonheme iron monooxygenases with N(1)- and N(3)-specific N-demethylation activity, respectively. Activity for both enzymes is dependent on electron transfer from NADH via a redox-center-dense Rieske reductase, NdmD. NdmD itself is a novel protein with one Rieske [2Fe-2S] cluster, one plant-type [2Fe-2S] cluster, and one flavin mononucleotide (FMN) per enzyme. All ndm genes are located in a 13.2-kb genomic DNA fragment which also contained a formaldehyde dehydrogenase. ndmA, ndmB, and ndmD were cloned as His(6) fusion genes, expressed in Escherichia coli, and purified using a Ni-NTA column. NdmA-His(6) plus His(6)-NdmD catalyzed N(1)-demethylation of caffeine, theophylline, paraxanthine, and 1-methylxanthine to theobromine, 3-methylxanthine, 7-methylxanthine, and xanthine, respectively. NdmB-His(6) plus His(6)-NdmD catalyzed N(3)-demethylation of theobromine, 3-methylxanthine, caffeine, and theophylline to 7-methylxanthine, xanthine, paraxanthine, and 1-methylxanthine, respectively. One formaldehyde was produced from each methyl group removed. Activity of an N(7)-specific N-demethylase, NdmC, has been confirmed biochemically. This is the first report of bacterial N-demethylase genes that enable bacteria to live on caffeine. These genes represent a new class of Rieske oxygenases and have the potential to produce biofuels, animal feed, and pharmaceuticals from coffee and tea waste.     

Chronic Effects of Xanthines on Levels of Central Receptors in Mice

1.Chronic ingestion of caffeine causes a significant increase in levels of A₁-adenosine, nicotinic and muscarinic receptors, serotonergic receptors, GABA_A receptors and L-type calcium channels in cerebral cortical membranes from mice NIH Swiss strain mice.2.Chronic theophylline and paraxanthine had effects similar to those of caffeine except that levels of L-type channels were unchanged. Chronic theobromine, a weak adenosine antagonist, and 1-isobutyl-3-methylxanthine (IBMX), a potent adenosine antagonist and phosphodiesterase inhibitor, caused only an increase in levels of A₁-adenosine receptors. A combination of chronic caffeine and IBMX had the same effects on receptors as caffeine alone. Chronic 3,7-dimethyl-1-propargylxanthine (DMPX), a somewhat selective A_2A-antagonist, caused only an increase in levels of A₁-adenosine receptors. Pentoxyfylline, an adenosine-uptake inhibitor inactive at adenosine receptors, had no effect on receptor levels or calcium channels.3.A comparison of plasma and brain levels of xanthines indicated that caffeine penetrated more readily and attained somewhat higher brain levels than theophylline or theobromine. Penetration and levels were even lower for IBMX, paraxanthine, DMPX, and pentoxyfylline.4.The results suggest that effective blockade of both A₁ and A_2A-adenosine receptors is necessary for the full spectrum of biochemical changes elicited by chronic ingestion of xanthines, such as caffeine, theophylline, and paraxanthine.     

INHIBITION OF 5''-NUCLEOTIDASE IN RAT BRAIN BY METHYLXANTHINES

—Rat brain 5′-nucleotidase (EC 3.1.3.5) is inhibited by methylxanthines such as theophylline. Inhibition of the 5′-nucleotidase by theophylline appears more efficient than the inhibition of cAMP phosphodiesterase by this drug. A similar inhibition is observed with caffeine, theobromine, 7′-methyl-xanthine and 1-methylxanthine.     

Two Distinct Pathways for Metabolism of Theophylline and Caffeine Are Coexpressed in Pseudomonas putida CBB5

Pseudomonas putida CBB5 was isolated from soil by enrichment on caffeine. This strain used not only caffeine, theobromine, paraxanthine, and 7-methylxanthine as sole carbon and nitrogen sources but also theophylline and 3-methylxanthine. Analyses of metabolites in spent media and resting cell suspensions confirmed that CBB5 initially N demethylated theophylline via a hitherto unreported pathway to 1- and 3-methylxanthines. NAD(P)H-dependent conversion of theophylline to 1- and 3-methylxanthines was also detected in the crude cell extracts of theophylline-grown CBB5. 1-Methylxanthine and 3-methylxanthine were subsequently N demethylated to xanthine. CBB5 also oxidized theophylline and 1- and 3-methylxanthines to 1,3-dimethyluric acid and 1- and 3-methyluric acids, respectively. However, these methyluric acids were not metabolized further. A broad-substrate-range xanthine-oxidizing enzyme was responsible for the formation of these methyluric acids. In contrast, CBB5 metabolized caffeine to theobromine (major metabolite) and paraxanthine (minor metabolite). These dimethylxanthines were further N demethylated to xanthine via 7-methylxanthine. Theobromine-, paraxanthine-, and 7-methylxanthine-grown cells also metabolized all of the methylxanthines mentioned above via the same pathway. Thus, the theophylline and caffeine N-demethylation pathways converged at xanthine via different methylxanthine intermediates. Xanthine was eventually oxidized to uric acid. Enzymes involved in theophylline and caffeine degradation were coexpressed when CBB5 was grown on theophylline or on caffeine or its metabolites. However, 3-methylxanthine-grown CBB5 cells did not metabolize caffeine, whereas theophylline was metabolized at much reduced levels to only methyluric acids. To our knowledge, this is the first report of theophylline N demethylation and coexpression of distinct pathways for caffeine and theophylline degradation in bacteria.Caffeine (1,3,7-trimethylxanthine) and related methylxanthines are widely distributed in many plant species. Caffeine is also a major human dietary ingredient that can be found in common beverages and food products, such as coffee, tea, and chocolates. In pharmaceuticals, caffeine is used generally as a cardiac, neurological, and respiratory stimulant, as well as a diuretic (3). Hence, caffeine and related methylxanthines enter soil and water easily through decomposed plant materials and other means, such as effluents from coffee- and tea-processing facilities. Therefore, it is not surprising that microorganisms capable of degrading caffeine have been isolated from various natural environments, with or without enrichment procedures (3, 10). Bacteria use oxidative and N-demethylating pathways for catabolism of caffeine. Oxidation of caffeine by a Rhodococcus sp.-Klebsiella sp. mixed-culture consortium at the C-8 position to form 1,3,7-trimethyluric acid (TMU) has been reported (8). An 85-kDa, flavin-containing caffeine oxidase was purified from this consortium (9). Also, Mohapatra et al. (12) purified a 65-kDa caffeine oxidase from Alcaligenes sp. strain CF8. Cells of a caffeine-degrading Pseudomonas putida strain (ATCC 700097) isolated from domestic wastewater (13) showed a fourfold increase in a cytochrome P450 absorption spectrum signal compared to cells grown on glucose. Recently, we reported a novel non-NAD(P)⁺-dependent heterotrimeric caffeine dehydrogenase from Pseudomonas sp. strain CBB1 (20). This enzyme oxidized caffeine to TMU stoichiometrically and hydrolytically, without producing hydrogen peroxide. Further metabolism of TMU has not been elucidated.Several caffeine-degrading bacteria metabolize caffeine via the N-demethylating pathway and produce theobromine (3,7-dimethylxanthine) or paraxanthine (1,7-dimethylxanthine) as the initial product. Theophylline (1,3-dimethylxanthine) has not been reported to be a metabolite in bacterial degradation of caffeine. Subsequent N demethylation of theobromine or paraxanthine to xanthine is via 7-methyxanthine. Xanthine is further oxidized to uric acid by xanthine dehydrogenase/oxidase (3, 10). Although the identities of metabolites and the sequence of metabolite formation for caffeine N demethylation are well established, there is very little information on the number and nature of N-demethylases involved in this pathway.The lack of adequate information on the metabolism and enzymology of theophylline, caffeine, and related methylxanthines prompted us to investigate the degradation of these compounds in detail. We isolated a unique caffeine-degrading bacterium, P. putida CBB5, from soil via enrichment with caffeine as the sole source of carbon and nitrogen. Here we describe a detailed study of the metabolism of theophylline, caffeine, and related di- and monomethylxanthines by CBB5. Our results indicate that CBB5 initially N demethylated caffeine to produce theobromine (major product) and paraxanthine (minor product) before the pathways converged to 7-methylxanthine and xanthine. Surprisingly, CBB5 was also capable of utilizing theophylline as a sole carbon and nitrogen source. CBB5 N demethylated theophylline to 1-methylxanthine and 3-methylxanthine, which were further N demethylated to xanthine. Theophylline N-demethylase activity was detected in cell extracts prepared from theophylline-grown CBB5 cells. 1-Methylxanthine and 3-methylxanthine were detected as products of this NAD(P)H-dependent reaction. To our knowledge, this is the first report of a theophylline degradation pathway in bacteria and coexpression of distinct caffeine and theophylline degradation pathways.     

Potentiating effects of caffeine on teratogenicity of alkylating agents in mice

Teratogenic to subteratogenic doses of x-ray, mitomycin C, MNNG, thio-TEPA, cyclophosphamide, and chlorambucil were administered to pregnant ICR mice together with caffeine at doses of 12.5, 25, or 50 mg/kg on day 11 of gestation. Fetuses were examined for gross malformations on day 18 of gestation. The teratogenicity of mitomycin C was significantly potentiated by caffeine at a dose as low as 12.5 mg/kg. The teratogenicity of chlorambucil was also significantly potentiated by caffeine at 50 mg/kg, but similar potentiation was not observed for x-ray, MNNG, thio-TEPA, and cyclophosphamide.     

Coadministration of methylxanthines and inhibitor compounds potentiates teratogenicity in Xenopus embryos

Inhibitors of DNA synthesis (hydroxyurea and cytosine arabinoside), protein synthesis (cycloheximide and emetine), and nucleic acid synthesis (5-fluorouracil) were administered with each of three methylxanthines (caffeine, theophylline, and theobromine) to determine if teratogenic effects could be potentiated in Xenopus laevis embryos. The animals were exposed for 96 hours to methylxanthine and inhibitor concentrations that, alone, produced low percentages of malformations. Coadministration of caffeine or theophylline with each inhibitor greatly increased the incidence of malformed embryos. Similar potentiation was induced when theobromine and the protein synthesis inhibitors were tested. A lesser potentiative response was produced when theobromine and the nucleic acid synthesis inhibitor were administered together. Teratogenic potentiation did not occur when theobromine was administered in conjunction with the DNA synthesis inhibitors. Growth reduction in the treatments proved to be the most sensitive indicator of the potentiative effects. This study had two significant findings: the teratogenicity of the protein synthesis inhibitors was greatly increased upon coadministration with each methylxanthine, even though they are typically not very teratogenic by themselves, and coadministration of the DNA synthesis inhibitors with theobromine did not result in teratogenic potentiation. Additionally, this study serves as one method of validating the frog embryo teratogenesis assay-Xenopus (FETAX), since the results obtained concur with results from similar mammalian studies.     

Prevention of caffeine-induced limb malformations by maternal adrenalectomy

Caffeine at high doses is a known rodent teratogen and induces limb malformations along with cleft palate in various strains of rats and mice. Fujii and Nishimura ('74) postulated that caffeine was teratogenic by virtue of catecholamine release from maternal or embryonic tissue. We tested this hypothesis by surgically removing the maternal adrenal gland on day 6 of pregnancy and then administering 175 mg/kg of caffeine intraperitoneally at 1600 h day 11 and 900 h day 12. The teratogenic effects of caffeine in adrenalectomized versus nonadrenalectomized AKR mice were assessed in day 18 fetuses. Thirty percent of the surviving offspring were malformed in caffeine-treated, nonadrenalectomized dams compared to 7% of the offspring from adrenalectomized dams. Therefore we believe caffeine teratogenesis is initiated by release of catecholamines from the maternal adrenal gland.     

Engineering a microbial platform for de novo biosynthesis of diverse methylxanthines

Engineered microbial biosynthesis of plant natural products can support manufacturing of complex bioactive molecules and enable discovery of non-naturally occurring derivatives. Purine alkaloids, including caffeine (coffee), theophylline (antiasthma drug), theobromine (chocolate), and other methylxanthines, play a significant role in pharmacology and food chemistry. Here, we engineered the eukaryotic microbial host Saccharomyces cerevisiae for the de novo biosynthesis of methylxanthines. We constructed a xanthine-to-xanthosine conversion pathway in native yeast central metabolism to increase endogenous purine flux for the production of 7-methylxanthine, a key intermediate in caffeine biosynthesis. Yeast strains were further engineered to produce caffeine through expression of several enzymes from the coffee plant. By expressing combinations of different N-methyltransferases, we were able to demonstrate re-direction of flux to an alternate pathway and develop strains that support the production of diverse methylxanthines. We achieved production of 270 μg/L, 61 μg/L, and 3700 μg/L of caffeine, theophylline, and 3-methylxanthine, respectively, in 0.3-L bench-scale batch fermentations. The constructed strains provide an early platform for de novo production of methylxanthines and with further development will advance the discovery and synthesis of xanthine derivatives.     

Threshold level for theophylline in doping analysis

HPLC in the reversed-phase mode is used to assay methylxanthines including theobromine, paraxanthine, theophylline and caffeine in urine. The calibration graphs show good linearity in the concentration range 0–10 μg/ml. The limit for accurate quantitation of theophylline was 0.25 μg/ml. Between 6 and 20% of the parent drug is recovered in urine (0–12 h) after the oral administration of sustained release preparations containing 150 and 250 mg theophylline to four volunteers. Theophylline levels above 0.25 μg/ml were found in 1539 out of 3885 urine samples collected from athletes during unannounced doping control in Flanders. Statistical evaluation of the results gives a far outside value [75th percentile + (3× interquartile range)] of 2.25 μg/ml. The ratio theophylline paraxanthine (TP/PX) as an indicator for the non-dietary intake of theophylline seems to be more reliable. The far outside ratio was 0.20. To ensure with the greatest possible degree of certainty that no false-positive result is reported, decision limits of 5 μg/ml and 0.50, for theophylline and the ratio TP/PX respectively, are proposed.     

Caffeine biosynthesis in young leaves of Camellia sinensis: In vitro studies on N-methyltransferase activity involved in the conversion of xanthosine to caffeine

The aim of this study was to investigate the S -adenosylmethionine dependent N -methyltransferase(s) (NMT) associated with the three methylation steps in the caffeine biosynthesis pathway in tea ( Camellia sinensis L.). NMT activity in cell-free preparations from young leaves was purified by anion-exchange and gel-filtration column chromatography. In both systems, a single zone of NMT activity, with broad substrate specificity was detected. The N-3 position of dimethylxanthine and monomethylxanthines was methylated more readily than N-1 while comparatively little substitution occurred at the N-7 locus. When xanthosine was used as a substrate only the N-7 position was methylated. These results indicate that a single NMT may participate in the conversion of xanthosine to caffeine. The apparent M_r of the NMT, estimated by gel filtration chromatography, was 61 000. The substrate specificity of the NMT is compatible with the operation of a xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine pathway as the main biosynthetic route to caffeine in young tea leaves. The data also indicate that the conversion of 7-methylxanthine → paraxanthine → caffeine may function as one of a number of minor pathways that also contribute to the production of caffeine.     

Caffeine biosynthesis in young leaves of Camellia sinensis: In vitro studies on N-methyltransferase activity involved in the conversion of xanthosine to caffeine

The aim of this study was to investigate the S -adenosylmethionine dependent N -methyltransferase(s) (NMT) associated with the three methylation steps in the caffeine biosynthesis pathway in tea ( Camellia sinensis L.). NMT activity in cell-free preparations from young leaves was purified by anion-exchange and gel-filtration column chromatography. In both systems, a single zone of NMT activity, with broad substrate specificity was detected. The N-3 position of dimethylxanthine and monomethylxanthines was methylated more readily than N-1 while comparatively little substitution occurred at the N-7 locus. When xanthosine was used as a substrate only the N-7 position was methylated. These results indicate that a single NMT may participate in the conversion of xanthosine to caffeine. The apparent M_r of the NMT, estimated by gel filtration chromatography, was 61 000. The substrate specificity of the NMT is compatible with the operation of a xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine pathway as the main biosynthetic route to caffeine in young tea leaves. The data also indicate that the conversion of 7-methylxanthine → paraxanthine → caffeine may function as one of a number of minor pathways that also contribute to the production of caffeine.     

Potentiating effect of caffeine on the teratogenicity of acetazolamide in C57BL/6J mice

Pregnant C57BL/6J mice were treated with 0 or 50 mg of caffeine (CAFF) per kg, and 0, 200 mg/kg (L) or 1,000 mg/kg (H) of acetazolamide (ACZM) during day 9 of gestation (9DPC). Individual fetuses were examined for gross morphological abnormalities and skeletal variations. The increase in fetal malformations seen, especially right forelimb electrodactyly, was augmented at both dose levels of acetazolamide by concomitant exposure to caffeine. Both frequency and severity of ectrodactyly were potentiated by caffeine. Skeletal examination revealed a reduction of the number of ossified cervical and caudal vertebral centra among litters exposed to ACZM at either dose. In either case (ACZM-H, ACZM-L) that effect was augmented by co-administration of CAFF. The first cervical vertebra (C1) appeared to provide the most sensitive index of teratogenic exposure. This study provides evidence that a subteratogenic dose of caffeine can potentiate the teratogenic effect of acetazolamide in C57BL/6J mice when dams are treated on day 9 of gestation. In addition, skeletal examination provided evidence that simultaneous treatment with both agents delayed fetal development. Many litters exposed to ACZM or both agents displayed a reduction in skeletal ossification even in the absence of gross morphological abnormalities, suggesting that ossification can be used as an indicator of prenatal exposure to potentially harmful substances in the C57BL/6 mouse strain.     

Theobromine Inhibits Uric Acid Crystallization. A Potential Application in the Treatment of Uric Acid Nephrolithiasis

Purpose

To assess the capacity of methylxanthines (caffeine, theophylline, theobromine and paraxanthine) to inhibit uric acid crystallization, and to evaluate their potential application in the treatment of uric acid nephrolithiasis.

Materials and Methods

The ability of methylxathines to inhibit uric acid nucleation was assayed turbidimetrically. Crystal morphology and its modification due to the effect of theobromine were evaluated by scanning electron microscopy (SEM). The ability of theobromine to inhibit uric acid crystal growth on calculi fragments resulting from extracorporeal shock wave lithotripsy (ESWL) was evaluated using a flow system.

Results

The turbidimetric assay showed that among the studied methylxanthines, theobromine could markedly inhibit uric acid nucleation. SEM images showed that the presence of theobromine resulted in thinner uric acid crystals. Furthermore, in a flow system theobromine blocked the regrowth of post-ESWL uric acid calculi fragments.

Conclusions

Theobromine, a natural dimethylxanthine present in high amounts in cocoa, acts as an inhibitor of nucleation and crystal growth of uric acid. Therefore, theobromine may be clinically useful in the treatment of uric acid nephrolithiasis.     

Reversed-phase liquid chromatographic investigation of UV-absorbing low-molecular-weight compounds in saliva

The reversed-phase mode of high-performance liquid chromatography was used to determine the intra- and inter-individual levels of UV-absorbing low-molecular-weight compounds in saliva. Many of the compounds known to occur in serum were also found in saliva; however, concentrations in saliva are lower. Both the intra- and inter-individual levels of these compounds vary significantly; in most cases, the inter-individual variance is 2–3 times the intra-individual variance.

Caffeine and its metabolites in saliva are also reported. A greater number of metabolites were found in the saliva of habitual coffee drinkers. After caffeine was administered orally, paraxanthine, theobromine, theophylline, 1-methylxanthine, and 1-methyluric acid were found in the saliva of an individual who did not drink coffee regularly. In this subject, the serum half-life for caffeine was 3.49 h and the saliva half-life was 3.27 h. The half-life of caffeine in an habitual coffee drinker who had refrained from caffeine products for four days was 4.39 h.     

Teratogenic action of platinum thymine blue

The teratogenic activity of the antitumor agent cisplatinum-2-thymine (platinum thymine blue) was investigated in rats. Pregnant Wistar-derived albino rats were given single ip injections of an aqueous solution of platinum thymine blue (PTB) at one day of pregnancy from day 5 through day 14 (sperm day=day 0). The dosages used ranged from 20 to 80 mg/kg maternal body weight. At autopsy (day 20) fetuses were recovered and subsequently examined for skeletal and soft-tissue abnormalities. PTB was embryolethal and teratogenic at several stages during rat gestation. Embryonic deat occured following all doses, and was dose dependent, except at day 5. The majority of malformed fetuses, however, were observed only after treatment at day 6 or 7 following injection with 50, 60, or 80 mg/kg. Eye defects were the predominant abnormality followed by hydrocephalus, gastroschisis, and ectopia cordis. The skeleton was only slightly affected. PTB is a potent inhibitor of DNA synthesis, but its mechanism of teratogenic action is unknown.