Effects of caffeine on human health.

Caffeine is probably the most frequently ingested pharmacologically active substance in the world. It is found in common beverages (coffee, tea, soft drinks), in products containing cocoa or chocolate, and in medications. Because of its wide consumption at different levels by most segments of the population, the public and the scientific community have expressed interest in the potential for caffeine to produce adverse effects on human health. The possibility that caffeine ingestion adversely affects human health was investigated based on reviews of (primarily) published human studies obtained through a comprehensive literature search. Based on the data reviewed, it is concluded that for the healthy adult population, moderate daily caffeine intake at a dose level up to 400 mg day(-1) (equivalent to 6 mg kg(-1) body weight day(-1) in a 65-kg person) is not associated with adverse effects such as general toxicity, cardiovascular effects, effects on bone status and calcium balance (with consumption of adequate calcium), changes in adult behaviour, increased incidence of cancer and effects on male fertility. The data also show that reproductive-aged women and children are 'at risk' subgroups who may require specific advice on moderating their caffeine intake. Based on available evidence, it is suggested that reproductive-aged women should consume

Analysis of sequence specificity of 5-bromodeoxyuridine-induced reversion in cells containing multiple copies of a mutant gpt gene.

For studies on molecular mechanisms of mutagenesis, it would be advantageous to transfer mutant genes with specific alterations into mammalian cells and use the transformed cells in reversion analyses. In the present paper, we describe an efficient method for analyzing reversion events occurring in cells that possess multiple copies of a mutational target gene. This method involves amplification of the chromosomally integrated target genes with the polymerase chain reaction (PCR) and restriction endonuclease digestion of the amplified product. Single reversion events that either create or destroy restriction endonuclease recognition sequences that encompass the site of the original mutation can be identified in a background of 10-20 copies of the gene that retain the mutant sequence. Using this method, we have analyzed revertants induced by 5-bromodeoxyuridine (BrdU) in a Chinese hamster ovary cell line that possesses multiple copies of a mutant bacterial gpt gene containing a specific alteration. The results of this study not only demonstrate the effectiveness of this method for analyzing reversion of a single gene copy in transfectants possessing multiple copies of a mutant target gene, but also demonstrate that the sequence specificity for BrdU-induced mutations is the same in Chinese hamster cells as previously observed with mouse cells.

Induction of terminal differentiation-resistant epidermal cells in mouse skin and in papillomas by different initiators during two-stage carcinogenesis.

Carcinogen treatment of normal mouse epidermal cells causes some cells, if cultured under the appropriate conditions, to continue to proliferate instead of terminally differentiate, forming foci at 37 degrees C in medium with a calcium level above 0.1 mM. We have examined these Calcium (Ca)-resistant cells formed in the skin of SENCAR mice after treatment with the carcinogen initiator 7,12-dimethylbenz[a]anthracene (DMBA) followed by tumor promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Although in our previous studies TPA promotion initially increased the size but reduced the number of foci caused by the carcinogen initiator N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), TPA promotion of DMBA-treated mice increased the size but had no effect on the number of foci. Papillomas resulting from DMBA plus TPA treatment contained many rapidly growing Ca-resistant cells, corroborating our earlier results with MNNG. Permanent cell lines prepared from papilloma-derived foci formed squamous cell carcinomas in nude mice after relatively short periods in culture. These data provide further evidence that Ca-resistant cells may be papilloma (and perhaps carcinoma) precursors in vivo. In addition, since TPA tends to reduce the number of early Ca-resistant cells caused by MNNG but not by DMBA, this may at least partially explain why treatment with DMBA plus TPA is much more effective in producing papillomas in SENCAR mice than is treatment with MNNG plus TPA.

Acetic acid, a potent agent of tumor progression in the multistage mouse skin model for chemical carcinogenesis.

Acetic acid, a very weak tumor promoter in the multistage mouse skin model for chemical carcinogenesis, was found to be very effective at enhancing cancer development, when applied during the progression phase of the model. Papilloma-bearing mice when repeatedly treated with acetic acid had a greater carcinoma incidence and a greater conversion of papillomas to carcinomas than vehicle treated mice. Selective cytotoxicity is discussed as a possible mechanism.

Effect of exogenous glutathione on tumor progression in the murine skin multistage carcinogenesis model.

Oxidative stress has been suggested to play an integral role in the cancer process. It may be particularly significant during tumor progression, where there is likely to be a large amount of free radicals generated by infiltrating inflammatory cells and dying tumor cells. In order to test this hypothesis, a variety of free radical scavengers and antioxidants were assessed for their ability to inhibit tumor progression. The murine skin multistage carcinogenesis model was used to generate papillomas, which are a population of putative precancerous lesions. Various test agents were applied topically to papillomas in order to determine if they would decrease the incidence of the malignant lesion, squamous cell carcinoma. The agents tested included: reduced glutathione (GSH), butylated hydroxyanisole, vitamin E, copper(II) (3,5-diisopropylsalicylate)2, sodium benzoate, N-acetyl cysteine and disulfiram. Under the conditions of our experiments, only GSH and disulfiram inhibited tumor progression to a significant degree. Additional studies indicated that GSH prevented cancer development in a dose-dependent manner. Another experiment demonstrated that when papillomas received repeated topical applications of diethylmaleate, a GSH-depleting agent, tumor progression was enhanced. Collectively these data suggest that sufficient glutathione levels may be important in preventing cancer formation.

Sequential alterations in growth control and cell dynamics of rat hepatocytes in early precancerous steps in hepatocarcinogenesis.

This set of experiments is the second of a series designed to explore alterations in cell dynamics and growth control of new populations of hepatocytes that appear to play a role in the carcinogenic process induced in the liver by chemical carcinogens. This is part of an ongoing study of the biochemical and molecular basis for cancer development. A rat model for hepatocarcinogenesis, the resistant hepatocyte model, was chosen with its synchrony of several steps in the process. Carcinogenesis was initiated by the administration of a single necrogenic dose of diethylnitrosamine. Resistant hepatocytes so induced were stimulated to proliferate rapidly to form nodules by a mitogenic stimulus in the presence of a brief exposure to dietary 2-acetylaminofluorene sufficient to inhibit the proliferation of the majority of uninitiated hepatocytes, the nonresistant population. A small subset of these hepatocyte nodules, the persistent nodules, was examined at 2, 4, and 6 mo postinitiation. Duration of phases of the cell cycle, growth fraction, doubling time, cell death, and cell loss and the responses and subsequent recovery after the application of a strong mitogenic stimulus, partial hepatectomy, were measured. The first precancerous hepatocyte nodule, at 2 mo, showed a "normal" duration of phases of the cell cycle. The growth fractions were about 4,4, and 8% at 2, 4, and 6 mo, respectively, as compared to 0.4% in the surrounding hepatocytes. Accompanying the increased growth fractions were considerable levels of cell loss, measuring about 3% at 2 mo and 7% at 6 mo. At 6 mo, the hepatocyte nodule population, unlike the hepatocytes in the surrounding liver, shows a failure to return to its base-line level after stimulation of cell proliferation by partial hepatectomy. The results of this study have identified two new steps in the early precancerous phase of hepatocarcinogenesis relating to alterations in the control of cell proliferation and are consistent with the hypothesis that new and evolving cell populations may play an important role in the step-by-step carcinogenic process. These new populations appear to acquire alterations in growth control in a seriatim fashion, with retention of some "normal" properties.

Critical genetic determinants and molecular events in multistage skin carcinogenesis.

Carcinogenesis can be operationally and mechanistically divided into at least three major stages--initiation, promotion, and progression. Variations among stocks and strains of mice to susceptibility to multistage skin and liver carcinogenesis appear to be more related to alterations in tumor promotion than tumor initiation; however, the critical events have not been determined. In the mouse skin model the first stage is thought to involve the interaction of a tumor initiator with the genetic material of stem cells leading to an alteration in some aspect of growth control, differentiation, or both. The major effect of tumor promoters, regardless of the type, is the specific expansion of the initiated stem cells in the skin. This appears to occur by both direct and indirect mechanisms that involve the loss of glucocorticoid receptors, differentiation alterations, a direct growth stimulation of the initiated cells, or selective cytotoxicity. The progression stage is characterized by a high level of genetic instability that produces a number of chromosomal alterations. These changes may be responsible for the loss of the high-molecular-weight keratin proteins and filaggrin, increase in gamma-glutamyl-transpeptidase activity, and changes in oncogene expression in squamous cell carcinomas. We have found that a high percentage of squamous cell carcinomas have a trisomy in chromosome 2 that carries both src and abl genes and an increased expression of src and abl. We have also found increased Ha-ras on RNA expression in both papillomas and squamous cell carcinomas. We suggest that the genetic instability of the initiated cells is responsible for most observed changes during skin carcinogenesis.

Cell cycle kinetics of rat hepatocytes in early putative preneoplastic lesions in hepatocarcinogenesis.

This set of experiments is the first of a series designed to explore facets of cell proliferation of hepatocytes during the carcinogenic process induced in liver by chemical carcinogens. A rat model for hepatocarcinogenesis, the resistant hepatocyte model, was used. A major advantage of this model is the unusual degree of synchrony in the early steps. Carcinogenesis was initiated by the administration of a necrogenic dose of diethylnitrosamine. Resistant hepatocytes so induced were stimulated rapidly to proliferate by partial hepatectomy in the presence of a brief exposure to dietary 2-acetylaminofluorene sufficient to inhibit the proliferation of the majority of hepatocytes, the nonresistant population. Cell cycle parameters were measured in the early carcinogen-altered resistant hepatocyte populations and in regenerating hepatocytes. Growth fraction and doubling time were experimentally determined in the altered hepatocytes. The mean cell cycle length of the resistant cells was 38.6 hr, somewhat longer than that of regenerating hepatocytes, which was 33.6 hr. Most of the increase was due to a prolonged S phase which was 13.6 hr in the altered cell population as compared to 7.0 hr in hepatocytes in regenerating control liver. The hepatocytes in normal regenerating liver had a mean duration of 21.6 hr for G1 as compared to 20.4 hr for the altered hepatocytes and a G2 of 3.4 hr as compared to 3.0 hr for carcinogen-altered hepatocyte. M was assumed to be 1.6 hr in both populations. The growth fraction in the altered cell population was determined to be a minimum of 0.83, and the doubling time was about 45 hr. Thus, the resistant hepatocytes which represent an early putative preneoplastic population show, in general, a prolongation of the cell cycle, mostly due to a prolonged S phase.

Failure of glutathione to prevent liver cancer development in rats initiated with diethylnitrosamine in the resistant hepatocyte model.

This study was undertaken to observe whether the administration of reduced glutathione intragastrically to male Fischer 344 rats during the precancerous steps of liver carcinogenesis has any protective effect on the development of hepatocellular carcinoma. Hepatocyte nodules were induced in the liver with a single initiating dose of diethylnitrosamine followed by selection of resistant hepatocytes to generate nodules by a two week exposure to dietary 2-acetylaminofluorene coupled with partial hepatectomy. Animals had hepatocyte ('hyperplastic') nodules when examined by laparotomy at three months. At that time, the animals were divided into two groups. One received daily intragastric glutathione for 8 months while the other received no further treatment. An additional control group received only the selecting (promoting) regimen with no initiator or glutathione. At 12 months, the animals receiving the initiator and promoter regimen had a 65% incidence of hepatocellular carcinoma and those receiving glutathione in addition had a 71% incidence. Under these experimental conditions, the long term administration of glutathione appears to have no observable influence on liver cancer development in this model.