Cellular binding proteins for vitamin A in human carcinomas and in normal tissues.

Blinded analyses of the concentrations of binding proteins for retinol and retinoic acid (CRABP) in homogenates of cancer and normal tissue aliquots obtained from human cervix, endometrium, ovary, breast, and lung were carried out by the sucrose gradient ultracentrifugation technique. In carcinomas of the cervix and endometrium, CRABP mean values of 50.4 and 123.2 pmol/g tissue, respectively were detected. Such concentrations represent a 3- and 4-fold increase over the mean values of CRABP in the normal cervix (16.9 pmol/g) and normal endometrium (30.8 pmol/g), respectively. In carcinomas of the ovary, the mean CRABP level was 128.6 pmol/g compared to the maximal mean value of less than or equal to 0.46 pmol/g in the normal ovary. Elevated levels of CRABP were also found in breast and lung carcinomas compared to the amounts detected in the same patient in normal tissue aliquots of the same organ. The differences between CRABP concentrations in cervical, endometrial, ovarian, and breast carcinomas and those in normal tissue are statistically significant. In contrast, cellular retinol-binding protein concentrations were reduced in the endometrial, ovarian, breast, and lung carcinomas compared to normal tissues. There were no significant differences between the log-mean concentrations of cellular retinol-binding proteins in the cytosols from tissue aliquots of carcinoma of the cervix and those in the cytosols from tissue aliquots of normal cervix.

Cellular binding proteins for vitamin A in the normal human uterine cervix and in dysplasias.

Cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein are present in the cytosol of normal human uterine cervical tissues, as detected by ultracentrifugation analysis. Both binding proteins have characteristically high specificity for their respective ligands. In sucrose gradients, both proteins sediment in the 2S region and are of similar molecular weight (M.W. approximately 14,000). In blind analyses of cervical biopsies, obtained under direct vision by colposcopy of normal women (control) or from patients histopathologically diagnosed to have dysplasias or carcinoma in situ (study group), CRBP was not detectable by sucrose gradient analysis in 78.8% of the 33 abnormal biopsies, compared to 23.5% of the 34 controls. This difference was statistically significant (p less than 0.005). In biopsies in which CRBP was detected, the mean levels were 2.76 and 0.72 pmol/mg protein in the cytosol for the control and study groups, respectively. In some subjects from each group, cellular retinoic acid-binding protein but not CRBP was detected in the biopsied tissue. The presence and role of these binding proteins in vitamin A metabolism, epithelial maturation and differentiation in cervical dysplasias, and in situ lesions remain to be investigated.

Urinary cyclic nucleotides and the cytopathology of human uterine cervical dysplasias.

Blinded urinary assays for cyclic guanosine 3':5'-monophosphate (cGMP) and cyclic adenosine 3':5'-monophosphate (cAMP) were performed on 49 subjects with documented abnormal cervical cytology and 21 control subjects with normal cytology. A significant difference in the mean cGMP:cAMP ratios between the case and control groups was found. A significantly greater proportion of women with cytological abnormalities had a cGMP:cAMP ratio above the 0.2 level (p less than 0.001). Cases treated surgically for severe dysplasia or carcinoma in situ of the cervix revealed a significant postsurgical fall in the cGMP:cAMP ratios (p less than 0.025). The possibility of utilizing urinary ratios of cyclic nucleotides as an objective index in the detection, monitoring of progression, and therapy of preneoplastic cervical lesions is discussed.

Induction of transforming growth factor beta-1 in cervical intraepithelial neoplasia in vivo after treatment with beta-carotene.

Transforming growth factor (TGF) beta1 is a potent growth inhibitor of epithelial cells. Loss of responsiveness to TGF-beta1 and/or loss of TGF-beta1 itself may be important in the progression of cervical intraepithelial neoplasia to invasive cervical cancer. Retinoids have antiproliferative effects on epithelial cells and have been used as chemopreventive and chemotherapeutic agents for several human cancers. There is evidence that retinoids exert their effects by promoting the induction of TGF-beta. The aim of this study was to determine whether the expression of TGF-beta1 was altered in patients enrolled in a clinical trial designed to test the therapeutic efficacy of beta-carotene, a carotenoid metabolized to retinol, in cervical intraepithelial neoplasia. Using an immunohistochemical technique, tissues were stained with two types of antisera that react with the intracellular and extracellular forms of TGF-beta1. Matched cervical biopsies taken from 10 patients before and after treatment with beta-carotene were immunostained simultaneously to allow direct comparison of relative staining intensity. A significant increase in intracellular TGF-beta1 immunoreactivity was noted in cervical epithelial cells in patients with cervical intraepithelial neoplasia after treatment with beta-carotene (P = 0.003). These results demonstrate regulation of a TGF-beta isoform in vivo in humans in response to beta-carotene administered as a chemopreventive agent.

Plasma levels of beta-carotene, lycopene, canthaxanthin, retinol, and alpha- and tau-tocopherol in cervical intraepithelial neoplasia and cancer.

Epidemiological studies continue to identify an association of dietary antioxidant micronutrients in cancer prevention. A number of case-control and cohort studies have demonstrated a relationship between high intake of foods rich in carotenoids, tocopherols, and vitamin C with a reduced risk of certain human malignancies. The purpose of this study was to investigate the comparative plasma levels of a profile of known dietary antioxidants, namely, beta-carotene, lycopene, canthaxanthin, retinol, alpha-tocopherol, and tau-tocopherol. The target population was women with a histopathological diagnosis of cervical intraepithelial neoplasia (CIN) or cervical cancer and a control group. All women resided in the same catchment area (Bronx Borough, New York City) and were of similar inner-city socioeconomic backgrounds representing a fairly homogenous population group. A cross-sectional sample of 235 women was recruited with informed consent. Plasma nutrient levels were measured by reverse-phase high pressure liquid chromatography under study codes. The mean plasma levels of carotenoids (beta-carotene, lycopene, and canthaxanthin), as well as alpha-tocopherol, were significantly lower in women with CIN and cervical cancer. In contrast, the mean plasma level of tau-tocopherol was higher among patients with CIN, while the mean plasma level of retinol was comparable among the groups. There were significant linear trends for all three carotenoids and quadratic trends for alpha- and tau-tocopherol with the degree of cervical histopathology. Plasma beta-carotene concentrations in cigarette smokers were significantly lower regardless of cervical pathology, whereas plasma lycopene and canthaxanthin levels were significantly lower in smokers with CIN. The findings of a decrease in all plasma antioxidant nutrient levels except tau-tocopherol in women with CIN and cancer suggest a potential role for antioxidant deficiency in the pathogenesis of CIN and carcinoma of the cervix, which requires further investigation.

Sex difference in cellular retinol- and retinoic acid-binding proteins in human colon adenocarcinomas.

Human colon adenocarcinomas and adjacent non-cancerous, normal colon from the same patient were assayed for the presence and amounts of cellular binding proteins for retinol (CRBP) and retinoic acid (CRABP) by sucrose gradient analysis. In male patients, the mean concentrations of both CRBP and CRABP in the colon cancers were statistically significantly higher than in the adjacent normal colon. By contrast, in female colon cancers, the mean levels for both binding proteins were reduced approximately 2-fold, compared to the concentrations in the adjacent normal colon. These findings reveal an unexpected sex difference in the binding proteins for retinol and retinoic acid in human colon malignancies.

Placental and serum levels of carotenoids in preeclampsia.

OBJECTIVE: We compared placental tissue, maternal serum, and umbilical cord venous blood levels of four dietary carotenoids (alpha-carotene, beta-carotene, lycopene, and canthaxanthin) in normal pregnant women and those with preeclampsia. METHODS: Levels of alpha-carotene, beta-carotene, lycopene, and canthaxanthin were measured in placental tissue, maternal serum, and umbilical cord venous blood from 22 normal pregnant women and 19 women with preeclampsia. The criteria for recruitment included gestational age of 30-42 weeks, singleton pregnancy, intact membranes, absence of labor contractions, and absence of any other medical complication concurrent with preeclampsia. Carotenoids were measured using high-pressure liquid chromatography. RESULTS: All four carotenoids were detectable in human placental tissue, maternal serum, and umbilical cord venous blood samples. The levels of beta-carotene, lycopene, and canthaxanthin in placentas from preeclamptic women were significantly lower (P =.032, .009, and .013, respectively, by Mann-Whitney test) than those from normal pregnant women. Maternal serum levels of beta-carotene and lycopene were significantly lower (P =.004 and .008, respectively, by Mann-Whitney test) in women with preeclampsia. However, umbilical cord venous blood levels of these carotenoids were not significantly different between the two groups. CONCLUSION: Lower placental tissue and maternal serum carotenoid levels in women with preeclampsia suggest that oxidative stress or a dietary antioxidant influence might have an effect on the pathophysiology of preeclampsia.

Plasma concentrations of coenzyme Q10 and tocopherols in cervical intraepithelial neoplasia and cervical cancer.

Cervical intraepithelial neoplasia (CIN) may, at times, unpredictably progress to invasive carcinoma of the cervix. Epidemiological nutritional studies suggest that higher dietary consumption and circulating levels of certain micronutrients may be protective against cervical cancer. However, a preventive role of dietary antioxidants in CIN is not well established. The purpose of this cross-sectional study was to investigate the comparative plasma concentrations of three potent antioxidants, coenzyme Q(10,) alpha-tocopherol and gamma-tocopherol, in women with normal Pap smears and patients with a biopsy-confirmed histopathological lesion diagnosed as CIN or cervical cancer. Plasma concentrations of coenzyme Q(10,) alpha-tocopherol and gamma-tocopherol were measured by high-pressure liquid chromatography in both normal women without any history of abnormal Pap smears (n=48), and patients with histopathologically confirmed diagnoses of: (a) CIN I, n=98; (b) CIN II, n=49; (c) CIN III, n=10; and (d) cervical cancer, n=25. The mean plasma levels of coenzyme Q(10), alpha-tocopherol and gamma-tocopherol were significantly lower (P<0.001,<0.001, and<0.001, respectively by Kruskal-Wallis test) in patients with various grades of CIN and cervical cancer compared with controls. After controlling for age and smoking, an inverse association between histological grades of epithelial lesions and both plasma coenzyme Q(10) and alpha-tocopherol concentrations was observed. The low plasma concentrations of coenzyme Q(10) may be due to deficient dietary intake or a decrease in endogenous coenzyme Q(10) biosynthesis that may reflect increased utilization as a result of free radical reactive oxygen species induced oxidative stress. Further molecular studies on the mechanistic role of antioxidants in women with precancer cervical lesions are needed.

Plasma reduced and total ascorbic acid in healthy women: effects of smoking and oral contraception.

This is the first report of both reduced (RAA) and total (TAA) plasma ascorbic acid levels in women who smoke and are on oral contraception. 155 normal healthy subjects were investigated. A study questionnaire was completed including age, method of contraception, smoking status, and food intake at breakfast, prior to attending the clinic. Biochemical assays were promptly carried out without knowledge of subject's clinical or dietary status. One-way analysis of variance revealed that oral contraception per se, barrier or IUD methods have no effect on plasma ascorbic acid levels. The mean values (mg/dl +/- SD) for plasma RAA between smokers and nonsmokers were 0.512 +/- 0.241 and 0.601 +/- 0.263; and that for TAA were 0.565 +/- 0.232 and 0.682 +/- 0.231, respectively. Significant decreases in both plasma RAA (p less than 0.05) and TAA levels (p less than 0.001) were observed in smokers. Age was an interacting variable. No association of smoking or oral contraceptive use was seen with RAA or TAA levels among women less than 26 years, but decreases in both RAA and TAA levels were evident among smokers aged 26 years or older. The present study emphasizes the total exposure to smoking, e.g., pack years, is a significant confounding variable in the study of plasma ascorbic acid levels.

Specific effect of zinc ions on DNA polymerase activity of avian myeloblastosis virus.

The effect of selected cations on DNA synthesis by DNA-polymerase of avian myeloblastosis virus (AMV) was studied. Zinc ions at low concentration (0.2mM) in the assay system enhanced the activity about 2 x fold and at higher concentration (2.0 mM) inhibited the activity completely. In contrast, addition of lithium and potassium salts produced inhibitory effects in this ionic concentration range. Replacement of K+ ion had an inhibitory effect on the activity.

DNA content in human uterine cervical dysplasias.

The DNA content of nuclei extracted from separate biopsies of normal cervical epithelium and from dysplastic sites in the same patient was determined biochemically. Mean percentage increase in the DNA content of dysplasias (mild: 143.3 +/- 14.9; moderate: 225.8 +/- 51.1; and severe: 359.8 +/- 45.8) was found to be statistically significant (F-Test: p less than 0.001) over the normal values in control cervical tissues.

Decreased beta-carotene tissue levels in uterine leiomyomas and cancers of reproductive and nonreproductive organs.

The dietary importance of beta-carotene as a factor in health maintenance has recently attracted considerable interest. Previously, total carotene content was estimated in a limited number of human tissues by means of spectrophotometric methods. In this study the levels of beta-carotene were measured by high-pressure liquid chromatography in tissue samples of uterine leiomyomas and adjacent normal myometrium obtained at hysterectomy from uteri of 18 patients. beta-Carotene concentration was significantly (p = 0.0013) lower in fibroid tissue than in normal myometrium. In addition, levels of beta-carotene were assayed in tissue samples of cancers of the cervix, endometrium, ovary, breast, colon, lung, liver, and rectum and were compared with levels of respective adjacent normal sites. The concentrations of beta-carotene were found to be lower in all cancer tissues. The decreased levels of beta-carotene suggest that beta-carotene deficiency may have a role in the cause and/or pathogenesis of leiomyomas and cancers of the organs that were investigated. The mechanism of action, however, remains unknown.

Vaginal hydrolysis of retinyl acetate: increase in plasma retinol and retinol binding protein in women with cervical dysplasias.

We previously reported the clinical feasibility of a Phase I trial involving the topical administration of a RA gel applied cervicovaginally in women with mild or moderate cervical dysplasia. Now, we report hydrolysis and systemic absorption of the RA gel from the vagina. HPLC analysis of samples of residual gel obtained from the cervical canal after topical bolus application indicate that the RA undergoes prompt in vivo hydrolysis yielding retinol as a major metabolite. Venous blood samples of 41 subjects, who self-administered a RA gel, were analyzed for plasma retinol and RBP concentrations prior to and upon completion of a 7-day treatment course and upon return for follow-up examinations. An increase in both the concentrations of plasma retinol and RBP were detected after topical application of the RA gel. These elevated values receded after the gel administration was discontinued. No significant changes were observed in plasma retinol or RBP concentrations in placebo-treated subjects. The efficacy of RA as a chemopreventive agent in treating cervical dysplasias remains to be determined.

Plasma retinol-binding protein in human uterine cervical dysplasias and cancer.

Plasma concentrations of retinol-binding protein (RBP) were measured in a cross-sectional study of asymptomatic normal menstruating women (n = 94) who obtained Pap smears and participated in a double-blinded nutritional survey. Controls (n = 45) were women with negative cervical cytology, normal colposcopy and no known gynecologic pathology or dysfunction. Cases (n = 49) were subjects with abnormal cytology and colposcopically directed biopsy that established cervical epithelial dysplasias histopathologically in the previous 12-month period. In addition, 8 women with cancer of the cervix were also investigated. The mean plasma concentration of RBP was significantly lower in cases than in controls (p less than 0.001). In normal females, the follicular phase of the menstrual cycle was increased. This increase was absent in the plasma of patients with cervical dysplasias (p less than 0.05). The data direct attention to a possible etiologic association between RBP in human cervical epithelial abnormalities and cancer.

Plasma levels of antioxidant beta-carotene and alpha-tocopherol in uterine cervix dysplasias and cancer.

Chronic human health problems, namely arteriosclerosis, myocardial ischemia, and cancer, may be caused by highly active oxygen species and may be preventable by antioxidant vitamins. In humans, the sources of two major antioxidants, beta-carotene and alpha-tocopherol, are dietary. In this study, we measured the plasma concentrations of beta-carotene and alpha-tocopherol by reverse-phase high-pressure liquid chromatography in a cross-sectional sampling of 116 women. Significantly reduced plasma levels of beta-carotene and alpha-tocopherol were observed in women with histopathologically diagnosed cervical dysplasias or cancer (p less than 0.0001 and p less than 0.005, respectively). There was an inverse association between the plasma levels of both beta-carotene and alpha-tocopherol and increasingly severe graded cervical histopathology. In groups with advanced dysplasias, the percentage of smokers was markedly increased and the women were comparatively older (p less than 0.0001). A strong association was noted between smoking status and plasma beta-carotene levels, independent of cervical pathology. However, this was not evident with respect to alpha-tocopherol. The findings suggest that the antioxidants beta-carotene and alpha-tocopherol have biologic functions that are interdependent in the pathogenesis of cervical intraepithelial lesions and cervical cancer.

Effects of smoking and oral contraception on plasma beta-carotene levels in healthy women.

Oral contraceptive use and smoking have been known to affect plasma vitamin levels. Total carotenoids have been studied with spectrophotometry, a relatively insensitive technique. In this study plasma concentrations of beta-carotene and retinol were measured in coded samples by sensitive high-pressure liquid chromatography in a cross-sectional study of 149 normal healthy women attending a family planning clinic. At the time of recruitment in the morning, a general health questionnaire was administered for patient age, methods of contraception, smoking habits, and food intake at breakfast. Of the 149 enrolled volunteers, 88 were oral contraceptive users and 61 were not users. Among users, 21 smoked cigarettes, and there were 18 smokers among nonusers. Oral contraceptive users had significantly lower plasma concentrations of beta-carotene (p less than 0.001) and higher retinol levels (p less than 0.0001). Plasma beta-carotene or retinol levels did not differ among users of intrauterine contraceptive devices or barrier methods of contraception. No association was noted between the plasma levels of these two micronutrients and age greater than or less than 30 years. Cigarette smoking alone was associated with significantly reduced plasma beta-carotene levels in nonusers (p less than 0.001). Combined cigarette smoking and oral contraceptive usage were associated with low plasma beta-carotene levels; the results appear to be additive. These findings suggest a possible synergistic effect on plasma beta-carotene levels from the use of both cigarette smoking and oral contraception.

Lipid-soluble antioxidants: beta-carotene and alpha-tocopherol levels in breast and gynecologic cancers.

Free radical-induced damage is etiologically implicated in many chronic diseases including cancer. Epidemiologic data suggest an association between increased dietary intake of nutrients that are high in antioxidant vitamins and protection against the incidence of some human cancers. The purpose of this study was (a) to determine whether specific tissue antioxidants (beta-carotene and alpha-tocopherol) and any differences in their levels were measurable in randomly selected human breast and gynecologic malignant neoplasms and nonneoplastic tissue samples obtained from the same patient and (b) to establish normal ranges of these two antioxidant levels in human female reproductive tract tissues. Tissue samples were excised immediately from surgical specimens and released by staff pathologists from a spectrum of human female cancers. Neoplastic and adjacent nonneoplastic tissues samples were obtained from the same patient. Normal reproductive tract tissue samples were obtained from women undergoing hysterectomy for benign gynecologic conditions. Breast carcinoma and adjacent nonmalignant tissue specimens were obtained from women undergoing mastectomy. The concentrations of beta-carotene and alpha-tocopherol were measured by high-pressure liquid chromatography. In the same patient, beta-carotene levels were significantly lower in the cervical (P < 0.01) and endometrial (P < 0.005) carcinoma tissues than the levels detectable in adjacent nonneoplastic sites. In contrast, beta-carotene levels were higher in the ovarian (P < 0.05), breast (P < 0.005), and vulva (P < 0.05) carcinoma tissues. The alpha-tocopherol concentrations were significantly higher in the cancer tissues of cervix (P < 0.01) and endometrium (P < 0.001) than those in adjacent noninvolved tissue sites. The tissue concentrations of alpha-tocopherol in malignant and adjacent normal sites in breast, ovary, and vulva were comparable. For the first time, the ranges for beta-carotene and alpha-tocopherol levels in the normal female reproductive tract tissues were also established. The present findings of contrasting tissue levels of the antioxidants (beta-carotene and alpha-tocopherol) in breast, cervix, endometrium, ovary, and vulva cancers and in nonneoplastic tissues of the same patient suggest an organ-specific and heterogenous distribution. These antioxidants appear to be essential nutritional requirements of the human female reproductive tract and breast and are implicated in the pathophysiology and carcinogenesis of these human organs. The findings require further study of the role of these antioxidant nutrients in epithelial cell proliferation, maturation, and differentiation.

Smoking and the antioxidant ascorbic acid: plasma, leukocyte, and cervicovaginal cell concentrations in normal healthy women.

The influence of cigarette smoking on the plasma, leukocyte, and cervicovaginal cell ascorbic acid levels in 46 healthy smokers and healthy nonsmokers was investigated. Coded peripheral venous blood and cervicovaginal lavage specimens obtained after informed consent were analyzed simultaneously for their ascorbic acid content. The findings suggest that smoking affects the levels and distribution of ascorbic acid. In smokers (n = 16), the number of exfoliated cervicovaginal epithelial cells and leukocyte ascorbic acid levels was significantly higher (p less than 0.01, p less than 0.05, respectively) compared with nonsmokers (n = 30). In addition, cervicovaginal cell ascorbic acid, plasma reduced and total ascorbic acid levels were significantly lower (p less than 0.001, p less than 0.01, p less than 0.01, respectively). The exfoliated epithelial cell ascorbic acid levels in nonsmokers was fourfold greater than that of leukocytes. The implications of these findings within the context of free radical-induced cellular pertubations in smokers are discussed.