Toxicology and carcinogenesis studies of a nondecolorized [corrected] whole leaf extract of Aloe barbadensis Miller (Aloe vera) in F344/N rats and B6C3F1 mice (drinking water study).

BACKGROUND: Extracts from the leaves of the Aloe vera plant (Aloe barbadensis Miller) have long been used as herbal remedies and are also now promoted as a dietary supplement, in liquid tonics, powders or tablets, as a laxative and to prevent a variety of illnesses. We studied the effects of Aloe vera extract on rats and mice to identify potential toxic or cancer-related hazards. METHODS: We gave solutions of nondecolorized extracts of Aloe vera leaves in the drinking water to groups of rats and mice for 2 years. Groups of 48 rats received solutions containing 0.5%, 1% or 1.5% of Aloe vera extract in the drinking water, and groups of mice received solutions containing 1%, 2%, or 3% of Aloe vera extract. Similar groups of animals were given plain drinking water and served as the control groups. At the end of the study tissues from more than 40 sites were examined for every animal. RESULTS: In all groups of rats and mice receiving the Aloe vera extract, the rates of hyperplasia in the large intestine were markedly increased compared to the control animals. There were also increases in hyperplasia in the small intestine in rats receiving the Aloe vera extract, increases in hyperplasia of the stomach in male and female rats and female mice receiving the Aloe vera extract, and increases in hyperplasia of the mesenteric lymph nodes in male and female rats and male mice receiving the Aloe vera extract. In addition, cancers of the large intestine occurred in male and female rats given the Aloe vera extract, though none had been seen in the control groups of rats for this and other studies at this laboratory. CONCLUSIONS: We conclude that nondecolorized Aloe vera caused cancers of the large intestine in male and female rats and also caused hyperplasia of the large intestine, small intestine, stomach, and lymph nodes in male and female rats. Aloe vera extract also caused hyperplasia of the large intestine in male and female mice and hyperplasia of the mesenteric lymph node in male mice and hyperplasia of the stomach in female mice.

Effect of Aloe vera whole leaf extract on short chain fatty acids production by Bacteroides fragilis, Bifidobacterium infantis and Eubacterium limosum.

AIMS: To investigate the effect of Aloe vera whole leaf extract on pure and mixed human gut bacterial cultures by assessing the bacterial growth and changes in the production of short chain fatty acids. METHODS AND RESULTS: Bacteroides fragilis, Bifidobacterium infantis, and Eubacterium limosum were incubated with Aloe vera extracts [0%, 0.5%, 1%, 1.5% and 2%; (w/v)] for 24 and 48 h. Short chain fatty acids production was measured by gas chromatography/mass spectrometry analyses. A significant linear increase in growth response to Aloe vera supplementation was observed at 24 h for each of the bacterial cultures; however, only B. infantis and a mixed bacterial culture showed a significant positive linear dose response in growth at 48 h. In pure bacteria cultures, a significantly enhanced dose response to Aloe vera supplementation was observed in the production of acetic acid by B. infantis at 24 h and of butyric acid by E. limosum at 24 and 48 h. In the mixed bacterial culture, the production of propionic acid was reduced significantly at 24 and 48 h in a dose-dependent fashion, whereas butyric acid production showed a significant linear increase. CONCLUSIONS: The results indicated that Aloe vera possessed bacteriogenic activity in vitro and altered the production of acetic, butyric and propionic acids by micro-organisms selected for the study. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the study suggest that consumption of a dietary supplement, Aloe vera, may alter the production of short chain fatty acids by human intestinal microflora.

Abnormal folate metabolism and genetic polymorphism of the folate pathway in a child with Down syndrome and neural tube defect.

The association of neural tube defects (NTDs) with Down syndrome (trisomy 21) and altered folate metabolism in both mother and affected offspring provide a unique opportunity for insight into the etiologic role of folate deficiency in these congenital anomalies. We describe here the case of a male child with trisomy 21, cervical meningomyelocele, agenesis of corpus callosum, hydrocephaly, cerebellar herniation into the foramen magnum, and shallow posterior cranial fossa. Molecular analysis of the methylenetetrahydrofolate (MTHFR) gene revealed homozygosity for the mutant 677C-->T polymorphism in both the mother and child. The plasma homocysteine of the mother was highly elevated at 25.0 micromol/L and was associated with a low methionine level of 22.1 micromol/L. Her S-adenosylhomocysteine (SAH) level was three times that of reference normal women, resulting in a markedly reduced ratio of S-adenosylmethionine (SAM) to SAH and significant DNA hypomethylation in lymphocytes. The child had low plasma levels of both homocysteine and methionine and a reduced SAM/SAH ratio that was also associated with lymphocyte DNA hypomethylation. In addition, the child had a five-fold increase in cystathionine level relative to normal children, consistent with over-expression of the cystathionine beta synthase gene present on chromosome 21. We suggest that altered folate status plus homozygous mutation in the MTHFR gene in the mother could promote chromosomal instability and meiotic non-disjunction resulting in trisomy 21. Altered folate status and homozygous TT mutation in the MTHFR gene in both mother and child would be expected to increase the risk of neural tube defects. The presence of both trisomy 21 and postclosure NTD in the same child supports the need for an extended periconceptional period of maternal folate supplementation to achieve greater preventive effects for both NTD and trisomy 21.

Homocysteine metabolism in children with Down syndrome: in vitro modulation.

The gene for cystathionine beta-synthase (CBS) is located on chromosome 21 and is overexpressed in children with Down syndrome (DS), or trisomy 21. The dual purpose of the present study was to evaluate the impact of overexpression of the CBS gene on homocysteine metabolism in children with DS and to determine whether the supplementation of trisomy 21 lymphoblasts in vitro with selected nutrients would shift the genetically induced metabolic imbalance. Plasma samples were obtained from 42 children with karyotypically confirmed full trisomy 21 and from 36 normal siblings (mean age 7.4 years). Metabolites involved in homocysteine metabolism were measured and compared to those of normal siblings used as controls. Lymphocyte DNA methylation status was determined as a functional endpoint. The results indicated that plasma levels of homocysteine, methionine, S-adenosylhomocysteine, and S-adenosylmethionine were all significantly decreased in children with DS and that their lymphocyte DNA was hypermethylated relative to that in normal siblings. Plasma levels of cystathionine and cysteine were significantly increased, consistent with an increase in CBS activity. Plasma glutathione levels were significantly reduced in the children with DS and may reflect an increase in oxidative stress due to the overexpression of the superoxide dismutase gene, also located on chromosome 21. The addition of methionine, folinic acid, methyl-B(12), thymidine, or dimethylglycine to the cultured trisomy 21 lymphoblastoid cells improved the metabolic profile in vitro. The increased activity of CBS in children with DS significantly alters homocysteine metabolism such that the folate-dependent resynthesis of methionine is compromised. The decreased availability of homocysteine promotes the well-established "folate trap," creating a functional folate deficiency that may contribute to the metabolic pathology of this complex genetic disorder.

Polymorphisms in genes involved in folate metabolism as maternal risk factors for Down syndrome.

Down syndrome is a complex genetic and metabolic disorder attributed to the presence of three copies of chromosome 21. The extra chromosome derives from the mother in 93% of cases and is due to abnormal chromosome segregation during meiosis (nondisjunction). Except for advanced age at conception, maternal risk factors for meiotic nondisjunction are not well established. A recent preliminary study suggested that abnormal folate metabolism and the 677C-->T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene may be maternal risk factors for Down syndrome. The present study was undertaken with a larger sample size to determine whether the MTHFR 677C-->T polymorphism was associated with increased risk of having a child with Down syndrome. Methionine synthase reductase (MTRR) is another enzyme essential for normal folate metabolism. A common polymorphism in this gene was recently associated with increased risk of neural tube defects and might also contribute to increased risk for Down syndrome. The frequencies of the MTHFR 677C-->T and MTRR 66A-->G mutations were evaluated in DNA samples from 157 mothers of children with Down syndrome and 144 control mothers. Odds ratios were calculated for each genotype separately and for potential gene-gene interactions. The results are consistent with the preliminary observation that the MTHFR 677C-->T polymorphism is more prevalent among mothers of children with Down syndrome than among control mothers, with an odds ratio of 1.91 (95% confidence interval [CI] 1.19-3.05). In addition, the homozygous MTRR 66A-->G polymorphism was independently associated with a 2. 57-fold increase in estimated risk (95% CI 1.33-4.99). The combined presence of both polymorphisms was associated with a greater risk of Down syndrome than was the presence of either alone, with an odds ratio of 4.08 (95% CI 1.94-8.56). The two polymorphisms appear to act without a multiplicative interaction.

Increase in plasma homocysteine associated with parallel increases in plasma S-adenosylhomocysteine and lymphocyte DNA hypomethylation.

S-Adenosylmethionine and S-adenosylhomocysteine (SAH), as the substrate and product of essential cellular methyltransferase reactions, are important metabolic indicators of cellular methylation status. Chronic elevation of SAH, secondary to the homocysteine-mediated reversal of the SAH hydrolase reaction, reduces methylation of DNA, RNA, proteins, and phospholipids. High affinity binding of SAH to the active site of cellular methyltransferases results in product inhibition of the enzyme. Using a sensitive new high pressure liquid chromatography method with coulometric electrochemical detection, plasma SAH levels in healthy young women were found to increase linearly with mild elevation in homocysteine levels (r = 0.73; p < 0.001); however, S-adenosylmethionine levels were not affected. Plasma SAH levels were positively correlated with intracellular lymphocyte SAH levels (r = 0.81; p < 0.001) and also with lymphocyte DNA hypomethylation (r = 0.74, p < 0.001). These results suggest that chronic elevation in plasma homocysteine levels, such as those associated with nutritional deficiencies or genetic polymorphisms in the folate pathway, may have an indirect and negative effect on cellular methylation reactions through a concomitant increase in intracellular SAH levels.

Single-site methylation within the p53 promoter region reduces gene expression in a reporter gene construct: possible in vivo relevance during tumorigenesis.

It is not known whether transcriptional suppression by de novo methylation occurs within the promoter region of the p53 gene during multistage tumorigenesis. To address this question, in vivo alterations in the CpG methylation within the rat p53 promoter region were evaluated in control, preneoplastic, and tumor tissue during tumor progression using the folate/methyl-deficient model of hepatocarcinogenesis. Alterations in CpG methylation were found to be site-specific and to vary depending on the stage of carcinogenesis. To further explore the effect of site-specific methylation on p53 promoter activity, reporter gene constructs were prepared containing specifically methylated sites within the p53 promoter region, and the transcriptional activity in cultured mammalian cells was determined in a transient transfection assay. Relative to the unmethylated construct as a positive control, single-site methylation at nucleotide (nt) -450, which occurs 216 nt upstream from the 85-nt minimal promoter region, suppressed promoter activity by 85%. In contrast, single-site methylation at nt -179, which occurs within the minimal essential promoter region, suppressed activity by only 20%. The p53 promoter constructs containing the singly methylated CpG site at nt -450 were then reevaluated for processive changes in methylation status 48 h after transfection, during maximum suppression of promoter activity. Restriction analysis with methylation-sensitive enzymes revealed that de novo methylation had occurred after transfection at previously unmethylated sites. These findings suggest that nt -450 may constitute a critical site for initiation of de novo methylation and processive spreading of methylation associated with transcriptional inactivation of the p53 gene. Furthermore, the results suggest a possible alternative mechanism for the silencing of the p53 gene in tumors that do not have p53 mutations.

Measurement of plasma and intracellular S-adenosylmethionine and S-adenosylhomocysteine utilizing coulometric electrochemical detection: alterations with plasma homocysteine and pyridoxal 5'-phosphate concentrations.

BACKGROUND: The relative changes in plasma and intracellular concentrations of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) may be important predictors of cellular methylation potential and metabolic alterations associated with specific genetic polymorphisms and/or nutritional deficiencies. Because these metabolites are present in nanomolar concentrations in plasma, methods of detection generally require time-consuming precolumn processing or metabolite derivatization. METHODS: We used HPLC with coulometric electrochemical detection for the simultaneous measurement of SAM and SAH in 200 microL of plasma, 10(6) lymphocytes, or 10 mg of tissue. Filtered trichloroacetic acid extracts were injected directly into the HPLC system without additional processing and were eluted isocratically. RESULTS: The limits of detection were 200 fmol/L for SAM and 40 fmol/L SAH. In plasma extracts, the interassay CV was 3.4-5.5% and the intraassay CV was 2.8-5.6%. The analytical recoveries were 96.8% and 97.3% for SAM and SAH, respectively. In a cohort of healthy adult women with mean total homocysteine concentrations of 7.3 micromol/L, the mean plasma value was 156 nmol/L for SAM and 20 nmol/L for SAH. In women with increased homocysteine concentrations (mean, 12.1 micromol/L), plasma SAH, but not SAM, was increased (P <0.001), and plasma pyridoxal 5'-phosphate concentrations were reduced (P <0.001). Plasma SAM/SAH ratios were inversely correlated with homocysteine concentrations (r = 0.73; P <0.01), and the SAM/SAH ratio in plasma was directly correlated with the intracellular SAM/SAH ratio in lymphocytes (r = 0.70; P <0.01). CONCLUSIONS: Increased homocysteine in serum is associated with an increase in SAH and a decrease in the SAM/SAH ratio that could negatively affect cellular methylation potential. Accurate and sensitive detection of these essential metabolites in plasma and in specific tissues should provide new insights into the regulation of one-carbon metabolism under different nutritional and pathologic conditions.

Abnormal folate metabolism and mutation in the methylenetetrahydrofolate reductase gene may be maternal risk factors for Down syndrome.

BACKGROUND: Down syndrome, or trisomy 21, is a complex genetic disease resulting from the presence of 3 copies of chromosome 21. The origin of the extra chromosome is maternal in 95% of cases and is due to the failure of normal chromosomal segregation during meiosis. Although advanced maternal age is a major risk factor for trisomy 21, most children with Down syndrome are born to mothers <30 y of age. OBJECTIVE: On the basis of evidence that abnormal folate and methyl metabolism can lead to DNA hypomethylation and abnormal chromosomal segregation, we hypothesized that the C-to-T substitution at nucleotide 677 (677C-->T) mutation of the methylenetetrahydrofolate reductase (MTHFR) gene may be a risk factor for maternal meiotic nondisjunction and Down syndrome in young mothers. DESIGN: The frequency of the MTHFR 677C-->T mutation was evaluated in 57 mothers of children with Down syndrome and in 50 age-matched control mothers. Ratios of plasma homocysteine to methionine and lymphocyte methotrexate cytotoxicity were measured as indicators of functional folate status. RESULTS: A significant increase in plasma homocysteine concentrations and lymphocyte methotrexate cytotoxicity was observed in the mothers of children with Down syndrome, consistent with abnormal folate and methyl metabolism. Mothers with the 677C-->T mutation had a 2.6-fold higher risk of having a child with Down syndrome than did mothers without the T substitution (odds ratio: 2.6; 95% CI: 1.2, 5.8; P < 0.03). CONCLUSION: The results of this initial study indicate that folate metabolism is abnormal in mothers of children with Down syndrome and that this may be explained, in part, by a mutation in the MTHFR gene.

Uracil misincorporation, DNA strand breaks, and gene amplification are associated with tumorigenic cell transformation in folate deficient/repleted Chinese hamster ovary cells.

Clinical and experimental evidence has linked nutritional folic acid status to both anti- and procarcinogenic activity. Folate supplementation of normal cells appears to have a protective effect; however, folate supplementation of initiated cells may promote neoplastic progression. Given these considerations, the present series of experiments examines alterations in DNA metabolism and cumulative DNA lesions using an in vitro model of folate deprivation and repletion. DNA repair-deficient CHO-UV5 cells were cultured in Ham's F-12 medium or in custom-prepared Ham's F-12 medium lacking in folic acid, thymidine and hypoxanthine for a period of 18 days without cell passage. The results indicated that progressive folate and nucleotide depletion leads to a significant increase in the ratio of dUTP/dTTP and to the misincorporation of uracil into DNA. These alterations were accompanied by growth inhibition, DNA strand breaks, abasic sites and phenotypic abnormalities. After 14 days in culture, there was significant increase in gene amplification potential in the chronically folate-deficient cells, but no significant increase in anchorage-independent growth or in neoplastic transformation. Acute folate repletion of the deficient cells was used as a proliferative stimulus under conditions of dNTP pool imbalance and multiple lesions in DNA. A further increase in gene amplification was accompanied by anchorage-independent growth and neoplastic cell transformation as evidenced by aggressive tumor growth in Balb/c nu/nu mice. Using a sensitive in vitro model system, these results emphasize the essentiality of folic acid for de novo nucleotide synthesis and the integrity of the DNA. However, the in vivo relevance, especially in terms of tumorigenic potential, is not clear.

A new HPLC method for the simultaneous determination of oxidized and reduced plasma aminothiols using coulometric electrochemical detection.

A new method has been developed that is capable of providing a complete profile of the most common monothiols and disulfides present in plasma or tissue extracts. The method utilizes reversed phase ion-pairing high performance liquid chromatography coupled with coulometric electrochemical detection to simultaneously quantify free oxidized and reduced aminothiols or total aminothiols after chemical reduction. The method is extremely sensitive, with limits of detection in the 5 fmol/mL range for monothiols and 50 fmol/mL for dithiols. The interassay and intraassay coefficients of variation for total and free aminothiols ranged between 1.2 and 5.8%. The mean recoveries for total and plasma aminothiols ranged between 97.1 and 102.8%. The aminothiols are quantified directly, without derivatization, and include methionine, homocysteine, homocystine, cystathionine, cysteine, cystine, cysteinylglycine, and oxidized and reduced glutathione. Because a complete aminothiol profile of metabolites in both the remethylation (anabolic) and transulfuration (catabolic) pathways of homocysteine metabolism can be determined simultaneously, this new method should be useful in determining the metabolic etiology of homocysteinemia and in designing appropriate nutritional intervention strategies. Basic research applications of this method should lead to an increased understanding of the metabolic pathology of aminothiol imbalance.

Characterization of cellular response to silicone implants in rats: implications for foreign-body carcinogenesis.

Foreign-body (FB) carcinogenesis is a classic model of multistage tumour development in rodents. Previous studies have demonstrated that the physical characteristics of the implant, and not the chemical composition, are the critical determinants of tumour development. The recent controversy over silicone breast implants has raised questions regarding the potential carcinogenicity of lifetime tissue exposure to silicone products. The present study was designed to determine whether the inflammatory and fibrotic reactions associated with silicone implants are due to a non-specific foreign-body reaction or whether these responses reflect the unique chemical composition of silicone. F344 rats were implanted subcutaneously with one of three biomaterials: silicone elastomer (Group 1); impermeable cellulose acetate filters (Group 2, positive control); or porous cellulose acetate filters (Group 3, negative control). The silicone and cellulose implants of Groups 1 and 2 have been previously shown to induce fibrosarcomas in rodents, whereas the porous cellulose acetate implants of Group 3 have been shown to be non-carcinogenic. One week and two months after implantation, the pericapsular tissues were evaluated using histopathological and in situ immunohistochemical analyses. Endpoints included expression of leucocyte antigens CD4 (T helper/inducer), CD8 (T suppressor/cytotoxic) and CD11 b/c (macrophage), proliferating cell nuclear antigen (PCNA) as an indicator of proliferation, and in situ end-labelling (ISEL) of 3'OH DNA strand breaks as an indicator of DNA damage and apoptosis. The results indicated that the acute and chronic cellular responses to silicone (Group 1) were not different from impermeable cellulose filters (Group 2) of identical size and shape, suggesting that these responses were not unique to silicone. The inflammatory response to the carcinogenic cellulose and silicone implants (Groups 1 and 2) was attenuated and associated with the formation of a thick fibrotic capsule. In contrast, the porous cellulose filters (Group 3) induced a markedly different cellular response in which the inflammatory reaction was more extensive, prolonged and associated with minimal fibrosis. Within the fibrotic capsule surrounding the tumorigenic implants, but not the non-tumorigenic implants, cell proliferation and apoptotic cell death were increased and associated with persistent DNA strand breaks. Taken together, the results suggest that the micrometre-scale surface morphology of the implant determines the nature of the subsequent cellular response which may predispose to tumour development. Further, these studies serve to emphasize the critical importance of appropriate physical controls in studies designed to evaluate carcinogenic or autoimmune manifestations associated with silicone implants in order to rule out the contribution of the chronic foreign-body reaction.

Apoptosis and proliferation under conditions of deoxynucleotide pool imbalance in liver of folate/methyl deficient rats.

Weanling male F344 rats were fed either a semi-purified diet low in methionine and lacking in choline and folic acid (folate/methyl deficient) or a supplemented control diet for periods of 2, 5, 7 days, 3 weeks, and 9 weeks. Two days after initiating the folate/methyl deficient diet in weanling F344 rats, the incidence of apoptotic bodies, identified by in situ end-labeling of 3'-OH DNA strand breaks, was significantly increased in liver sections from the deficient rats. Apoptotic cell death was confirmed biochemically by an increase in nuclear Ca2+/Mg2+-dependent endonuclease activity that paralleled the increase in apoptotic bodies over the 9-week feeding period. There was no morphologic evidence of necrotic foci or necrosis-associated inflammatory response over the 9-week period. Confirming that cell turnover is chronically elevated in this model, the increase in apoptotic rate was accompanied by a sustained increase in the mitotic index (MI). The DNA repair-associated enzyme, poly(ADPribose) polymerase (PARP), was similarly elevated and was associated with significant decreases in the substrate for ADPribose polymer synthesis, nicotinamide adenine dinucleotide (NAD). Because folate metabolites are essential for de novo purine and thymidine biosynthesis, prolonged deficiency in folic acid can induce an imbalance in the deoxynucleotide precursors for DNA replication/repair and negatively affect the fidelity of DNA synthesis. Using an HPLC method, hepatic deoxyuridine triphosphate (dUTP) levels were increased at 3 and 9 weeks after initiation of the deficient diet and levels of thymidine triphosphate (dTTP) were reduced. An increase in dUTP/ dTTP ratio is consistent with a block in folate-dependent de novo thymidylate biosynthesis and may predispose to uracil misincorporation and DNA repair-related DNA strand breaks.