Enhanced DNA repair, immune function and reduced toxicity of C-MED-100, a novel aqueous extract from Uncaria tomentosa.

Female W/Fu rats were gavaged daily with a water-soluble extract (C-MED-100) of Uncaria tomentosa supplied commercially by CampaMed at the doses of 0, 5, 10, 20, 40 and 80 mg/kg for 8 consecutive weeks. Phytohemagglutinin (PHA) stimulated lymphocyte proliferation was significantly increased in splenocytes of rats treated at the doses of 40 and 80 mg/kg. White blood cells (WBC) from the C-MED-100 treatment groups of 40 and 80 mg/kg for 8 weeks or 160 mg/kg for 4 weeks were significantly elevated compared with controls (P < 0.05). In a human volunteer study, C-MED-100 was given daily at 5 mg/kg for 6 consecutive weeks to four healthy adult males. No toxicity was observed and again, WBC were significantly elevated (P < 0.05) after supplement. Repair of DNA single strand breaks (SSB) and double strand breaks (DSB) 3 h after 12 Gy whole body irradiation of rats were also significantly improved in C-MED-100 treated animals (P < 0.05). The LD50 and MTD of a single oral dose of C-MED-100 in the rat were observed to be greater than 8 g/kg. Although the rats were treated daily with U. tomentosa extracts at the doses of 10-80 mg/kg for 8 weeks or 160 mg/kg for 4 weeks, no acute or chronic toxicity signs were observed symptomatically. In addition, no body weight, food consumption, organ weight and kidney, liver, spleen, and heart pathological changes were found to be associated with C-MED-100 treatment.

Comparison of antitumor activity of declopramide (3-chloroprocainamide) and N-acetyl-declopramide.

Previous studies have suggested that some of the antitumor activity of declopramide (3-chloroprocainamide) could be due to its metabolites. One metabolite has been identified as N-acetyl-declopramide (N-acetyl-3-chloroprocainamide). The aim of this study is to investigate the bioactivity of N-acetyl-declopramide and to compare it with its parent compound. The data have shown that N-acetyl-declopramide inhibited tumor cell growth in vitro in HL60 and K562 cells, and in vivo in scid mice xenografted with a human brain astrocytoma (T24), which was evaluated after oral doses of 20 and 40 mg/kg given at 0, 24 and 48 hr +/- a single im dose of cisplatin (7.5 mg/kg). The action was presumably by inducing DNA strand breaks and apoptosis. No acute toxic symptoms and no body weight loss were observed. N-acetyl-declopramide given orally or im gave a similar drug level in mouse serum 30 minutes after administration (p > 0.05). It had a greater antitumor activity in vitro in HL60 or K562 cells and a similar efficacy of inhibiting tumor growth in vivo, when compared with declopramide. These data provided an explanation for the primary result obtained in this study, i.e. declopramide administered orally at 40 mg/kg gave the same efficacy of inhibiting tumor growth as im injection although oral administration had a lower bioavailability due to the formulation of N-acetyl-declopramide. Based on these data, it was concluded that the antitumor properties of declopramide administered orally were not compromised by metabolism to N-acetyl-declopramide because the latter also has strong antitumor properties.

Induction of apoptosis and inhibition of proliferation in human tumor cells treated with extracts of Uncaria tomentosa.

Growth inhibitory activities of novel water extracts of Uncaria tomentosa (C-Med-100) were examined in vitro using two human leukemic cell lines (K562 and HL60) and one human EBV-transformed B lymphoma cell line (Raji). The proliferative capacities of HL60 and Raji cells were strongly suppressed in the presence of the C-Med-100 while K562 was more resistant to the inhibition. Furthermore, the antiproliferative effect was confirmed using the clonogenic assay, which showed a very close correlation between C-Med-100 concentration and the surviving fraction. The suppressive effect of Uncaria tomentosa extracts on tumor cell growth appears to be mediated through induction of apoptosis which was demonstrated by characteristic morphological changes, internucleosomal DNA fragmentation after agarose gel electrophoresis and DNA fragmentation quantification. C-Med-100 induced a delayed type of apoptosis becoming most dose-dependently prominent after 48 hours of exposure. Both DNA single and double strand breaks were increased 24 hours after C-Med-100 treatment, which suggested a well-established linkage between the DNA damage and apoptosis. The induction of DNA strand breaks coupled to apoptosis may explain the growth inhibition of the tumor cells by Uncaria tomentosa extracts. These results provide the first direct evidence for the antitumor properties of Uncaria tomentosa extracts to be via a mechanism of selective induction of apoptosis.

Quality control program for storage of biologically banked blood specimens in the Malmö Diet and Cancer Study.

A biological bank has been developed to extend the biochemical and molecular research base for a prospective study on diet and cancer in the city of Malmo, Sweden. The study entered individuals 45-69 years of age, of which 30,382 individuals (45%) participated. Each individual entering the bank has stored samples of viable mononuclear leukocytes (MNLs; -140 degrees C) and granulocytes (GRANs; -80 degrees C) or buffy coats (-140 degrees C), erythrocytes (-80 degrees C), and plasma/serum (-80 degrees C). The bioassays developed to monitor the quality of storage conditions were: (a) viability and growth response to phytohemagglutinin for MNLs; (b) DNA strand breakage for GRANs; (c) NAD content for erythrocytes; and (d) thiol status for plasma/serum. The yield, purity, and storage conditions were all quality controlled, and the samples were determined to be of high standard after 137-190 weeks of storage. No differences in yield and purity were found in samples banked by different laboratory technicians. Growth responses of MNLs were severely reduced (90%) after 40 weeks of storage, which justified switching from the storage of purified MNLs and GRANs to the more cost-effective banking of buffy coats. We conclude that the quality of the banked material, based on the biochemical analysis done, indicate that the storage conditions are optimal at least up to 3.5 years, except for the growth response of MNLs.

Feasibility and quality of biological banking of human normal and tumor tissue specimens as sources of DNA for the Malmö Diet and Cancer Study.

Human tumor and normal tissue specimens, which were collected from autopsy material 1-6 days postmortem, were compared with similar tissue specimens collected within 2 h after surgical resection and transport to the pathology department. The end point criteria used to evaluate the quality of the specimens for biological banking purposes were the extractability and yield of high molecular weight DNA and UV absorption ratios at 260:280 after collection and immediate storage of the specimens at -80 degrees C. The data demonstrated that autopsy material was a quality source of DNA, although of not such high quality as surgical biopsy specimens <2 h after resection. The advantages of using autopsy material to supplement surgical specimen collection sent to pathology, as opposed to using specimen collection at surgery wards or formalin-fixed material, as sources of DNA are: (a) large amounts of tumor and normal tissues from a variety of organ sites can be obtained without regard to the patient's health status; (b) a higher percentage of retrieval of incident cases of cancer in prospective designed trials is more likely to be achieved; and (c) the extractable DNA is of sufficiently high enough quality to permit direct analyses by molecular hybridization and sequence methodologies.

DNA repair enhancement by a combined supplement of carotenoids, nicotinamide, and zinc.

Four volunteers were involved for 5 weeks of a baseline period, followed by 7 weeks of a combined supplementation of nicotinamide, zinc, and carotenoids (Nicoplex). Blood sampling and bioassays were carried out every week during the evaluation period. The supplementation of Nicoplex resulted in statistically significant increased resistance to DNA single-strand breaks induced by H2O2 (DNA retained on filter % from 46.7 +/- 1.9 to 59.4 +/- 4.3; p < 0.01), increased DNA repair 60 min after induction of damage (DNA retained on filter % from 74.6 +/- 4.8 to 88.3 +/- 4.2; p < 0.01), elevated poly (ADP-ribose) polymerase (PARP) activity (p < 0.05), and an increased proliferative response to phytohemagglutinin (PHA) (p < 0.05) when compared with the levels before supplementation. However, when the same subjects were supplemented with nicotinamide, zinc, and carotenoids together with another 17 nutrients or minerals, there were no changes in DNA damage, DNA repair, or proliferative response to PHA. Through the use of a rat model, DNA repair of splenocytes 3 h after 12 Gy whole-body irradiation was significantly enhanced in rats supplemented with Nicoplex for 6 weeks (p < 0.05) and 8 weeks (p < 0.01). Comparison of Nicoplex and its components administered separately revealed that there was an additive effect on DNA repair for both single- and double-strand breaks (both p < 0.05). On the basis of the results, it is hypothesized that the enhanced effect of combined supplement of nicotinamide, zinc, and carotenoids on DNA repair depends on their diversified mechanisms of action while multinutrient supplementation may compromise the effects by inhibitory interactions including uptake and absorption.

Hypochlorous acid/N-chloramines are naturally produced DNA repair inhibitors.

Human mononuclear leukocytes (HML) respond to oxidative DNA damage by activation of ADP ribosylation and initiation of DNA repair synthesis (i.e. unscheduled DNA synthesis, UDS), whereas neutrophils do not. When neutrophils are added to HML cultures in ratios up to 4:1 ADP ribosylation becomes inhibited to approximately 50-60%. The ability of neutrophils to inhibit HML ADP ribosylation was shown to be dependent on H2O2, chloride ions and myeloperoxidase, which in turn are factors known to govern HOCl and N-chloramine production by phagocytic cells. HOCl and a model N-chloramine, chloramine T, were shown to give a dose-dependent inhibition of DNA repair using four independent estimates, namely ADP ribosylation, UDS and the repair of DNA strand breaks estimated by nucleoid sedimentation and alkaline elution profiles. All the DNA repair measurements used on HML were inhibited approximately 70-80% by 100 microM doses of HOCl or chloramine T, which was considered a biologically relevant dose because: (i) viable neutrophils equal in concentration to those found in blood could easily produce 100 microM levels in short-term culture; (ii) 100 microM doses of these agents were not acutely cytotoxic judged by trypan blue stained cells after 30-60 min exposure and under the conditions used for assay, but yet they abolished 86-95% of the growth response of HML to phytohemagglutinin.

Oxidative stress induces DNA damage and inhibits the repair of DNA lesions induced by N-acetoxy-2-acetylaminofluorene in human peripheral mononuclear leukocytes.

Human mononuclear leukocytes were exposed to prooxidants such as H2O2, phorbol-12-myristate-13-acetate, and 4-nitroquinoline-N-oxide, and the effects on induction of DNA damage and repair were evaluated. ADP ribosylation was activated by prooxidant exposure and the response was bimodal with peaks of activation occurring at about 30 min and 4-5 h. Other evidence for prooxidant-induced DNA damage was provided by nucleoid sedimentation assays. Unscheduled DNA synthesis (UDS) was only slightly induced by prooxidant exposure which suggested that either the DNA lesions were repaired by a short patch mechanism involving little UDS, or the repair process was inhibited by prooxidant exposures, or some combination of both. This point was clarified by the fact that the repair of DNA lesions induced by N-acetoxy-2-acetylaminofluorene, an inducer of large patch DNA repair, was inhibited in a dose-dependent manner by exposure to H2O2 and the inhibition was dependent on ADP ribosylation. In contrast, the repair of DNA strand breaks induced by prooxidant exposures as identified above were complete within about 8 h and the repair was independent of ADP ribosylation. Both ADP ribosylation and N-acetoxy-2-acetylaminofluorene-induced UDS were shown to be up- and down-regulated by the redox state of human mononuclear leukocytes indicating a unique mechanism of cellular control over DNA repair.

Lower rates of thymidine incorporation into DNA of skin fibroblasts from patients with adenomatosis of the colon and rectum.

Thymidine incorporation into DNA was examined in confluent fibroblast cultures which had been further arrested by 7-9 days' growth on 0.5% serum-supplemented medium. There were 13 fibroblast lines from patients with adenomatosis of the colon and rectum (ACR) and seven control fibroblast strains from unaffected relatives and spouses of ACR patients. After 24 h an ACR fibroblast strain showed a decreased rate of unscheduled DNA synthesis (UDS), whereas no such effects were observed with the control fibroblast strain. It was shown that the difference in UDS rates between control and ACR fibroblast strains was not due to [3H]-dThd catabolism and/or disappearance in the culture medium after a 34-h labelling period. When the percent increases in N-acetoxy-2-acetylaminofluorene (NA-AAF)-induced UDS and in [3H]-dThd incorporation in the presence of 10 mM hydroxyurea were compared between 24 h and 34 h incubation, the 13 ACR fibroblast lines were significantly lower compared to the corresponding values calculated for the seven control fibroblast strains. The ACR fibroblasts also had significantly elevated levels of spontaneous chromosome aberrations compared to controls. Furthermore, the percent increase in NA-AAF-induced UDS was negatively correlated with the level of spontaneous chromosome aberrations. These data suggest that there is a genetic instability in the cells from ACR patients that can be estimated by both UDS and cytogenetic analyses.

Mutagen sensitivity, smoking habits and enzyme induction in healthy middle-aged men.

Unscheduled DNA synthesis (UDS) (excision-repair) of N-acetoxy-2-acetylaminofluorene (NA-AAF) damage to the DNA of human lymphocytes and levels of 3H-labeled NA-AAF bound to the DNA (carcinogen binding) of lymphocytes after 18 hr of culturing were measured in a consecutive subsample of healthy middle-aged males attending a multiphasic health screening program at the Department of Preventive Medicine in Malmö during 3 weeks in November-December 1981, and compared relative to their smoking habits, body weight, serum cholesterol, and gamma-glutamyltransferase levels as well as aryl hydrocarbon hydroxylase inducibility. This study group numbered 66 males and was uniform in sex, age, and investigation time. No case of significant arterial hypertension was present. The UDS and carcinogen binding results showed no correlation with the other factors measured, with the exception of smoking which was strongly (P less than 0.01) associated with increasing levels of both the UDS and carcinogen binding values. It is concluded that under ordinary circumstances smoking may represent the most important exogenous factor which may modulate risk to cardiovascular disease and cancer by influencing individual mutagen sensitivity.

The effects of smoking, hypertension and cardiovascular disease on the estimation of unscheduled DNA synthesis and covalent binding to DNA induced by N-acetoxy-2-acetylaminofluorene in resting human mononuclear leukocytes.

The levels of N-acetoxy-2-acetylaminofluorene (NA-AAF)-induced unscheduled DNA synthesis (UDS) and of NA-AAF binding to DNA have been determined in resting mononuclear leukocytes from individuals with various smoking habits, heart infarct patients and subjects diagnosed for hypertension. Age-matched and blood-pressure-controlled smokers (n = 99) had significantly elevated levels of NA-AAF-induced UDS and NA-AAF binding to DNA when compared to nonsmokers (n = 75) similarly corrected for age and blood pressure. Heart infarct patients without any history of risk factors, as well as diagnosed hypertensives with normalized blood pressure, were not significantly different from matched controls when assessed by the NA-AAF method. Our results support the theory that increased mutagen sensitivity is associated with smoking and high blood pressure but not with cardiovascular disease itself via some mechanism of genetic selection.

A genetic component of the variance of N-acetoxy-2-acetylaminofluorene-induced DNA damage in mononuclear leukocytes determined by a twin study.

The level of N-acetoxy-2-acetylaminofluorene (NA-AAF)-induced unscheduled DNA synthesis and the level of covalent binding of NA-AAF to DNA were determined in the mononuclear leukocytes of monozygotic and diazygotic twin pairs (n = 16 for each type). A statistically significant high degree of heritability was calculated for both parameters which, in turn, indicate genetic control of individual levels of induced DNA damage by NA-AAF.

The effect of an individual's blood pressure on the percentage of total 7,12-dimethylbenz[a] anthracene metabolites that bind covalently to DNA in cultured resting lymphocytes.

A method for the quantitative analysis of the percent metabolism that results in covalent binding of 7,12-dimethylbenz[a]anthracene (DMBA) to DNA in viable resting human lymphocytes is described. The inter- and intra-experimental reproducibility as judged by the coefficient of variation and examined in the same individual over a 3-month period was 31.4% and 13.9%, respectively. When the lymphocytes from 30 hypertensive individuals were exposed to 1 microM DMBA for 18 h, the percent of total DMBA metabolites that bind DNA covalently was correlated to the blood pressures of the patients at the time of sampling (r = 0.53, P less than 0.005). No influences on the data from the type or duration of hypertensive drug treatment could be statistically determined for this sample of hypertensive patients. It was concluded that high blood pressure is a strong determinant in predisposing lymphocytes to increased genetic risk from induced DNA damage and that this relationship is not statistically affected by hypertensive drug therapy.

N-acetoxy-2-acetylaminofluorene induced unscheduled DNA synthesis of granulopoietic precursor cells in chronic myeloid leukemia.

Studies on N-acetoxy-2-acetylaminofluorene (NA-AAF) induced unscheduled DNA synthesis (UDS) of granulopoietic cells were performed in patients with chronic myeloid leukemia (CML). Sequential studies were carried out in some patients. Both biochemical and autoradiographic methods demonstrated that [3H]dT was incorporated into nonreplicating DNA of immature granulopoietic cells after NA-AAF damage and the 2 methods significantly correlated to each other (r = 0.63, n = 19). NA-AAF induced DNA synthesis was lower for myeloblasts than promyelocytes and myelocytes. Biochemically determined NA-AAF induced UDS was higher for immature granulopoietic cells in blood than in marrow. Sequential studies on granulopoietic blood cells suggested that phases of accelerated leukocytosis in CML can be preceded by increases of NA-AAF induced UDS. Whether increases in NA-AAF induced UDS relates to an amplification of repair enzymatic capacity closely correlated to the cellular replication capacity, or whether it reflects an increased sensitivity to DNA damage induction and the consequences thereof, was not resolved in this study. Nevertheless these results are consistent with the hypothesis that increases in NA-AAF induced UDS signal the evolution during chronic phase CML of cell populations of increasing malignancy which escape growth control.

Interindividual variation in the responses of cultured human lymphocytes to exposure from DNA damaging chemical agents: interindividual variation to carcinogen exposure.

Human population variability to standardized doses of N-acetoxy-2-acetylaminofluorene (NA-AAF) and 7, 12-dimethylbenz(a) anthracene (DMBA) was determined in cultured lymphocytes by measuring (a) differential stimulation of unscheduled DNA synthesis after 1 h induction of DNA damage by 10 micrometer NA-AAF, (b) the level of NA-AAF induced chromosome aberrations remaining after 8 h of DNA-repair synthesis, and (c) the level of [3H]DMBA bound to DNA after 18 h incubation of resting lymphocytes in 5 micrometer DMBA. All 3 parameters indicated individual variation to carcinogen exposure and were correlated to the population differences in age, sex, blood pressure and mortality rates. Males always had a greater potential to accumulate DNA-damage than did females regardless of the sampled population. DNA-damage potentials increased with increasing age, blood pressure or mortality rates. There was always proportionally greater DNA-damage potentials in the males than in females. The in vitro response of mature granulocytes to a 10 micrometer NA-AAF dose, as estimated by [3H] thymidine incorporation from unscheduled DNA synthesis, was much lower than lymphocyte response. Nevertheless, individual variations in granulocyte NA-AAF induced unscheduled DNA synthesis paralleled the inter-individual fluctuations observed in the lymphocyte responses to NA-AAF.

Deoxyribonucleic acid repair synthesis: a new factor for defining high blood pressure.

1. The level of DNA repair synthesis has been compared in males diagnosed as having hypertension (diastolic blood pressure greater than or equal to 100 mmHg), males with slightly elevated blood pressure (greater than 95th percentile corrected for age) and males with normal or subnormal blood pressure (less than or equal to 30th percentile corrected for age). 2. The hypertensive males and the males with elevated blood pressure could not be distinguished from each other with respect to the levels of chemically induced repair synthesis, but both were significantly increased over the induced repair synthesis values of individuals with normal or low blood pressure. Since it has been shown previously that lymphocytes with high repair synthesis values also have increased levels of both carcinogen-DNA binding and chromosomal aberrations (Nordén, Scherstén, Thulin, Pero, Bryngelsson & Mitelman, 1975), the biological significance of this variable appears to be of value in discriminating between normal and high blood pressure groups.

High blood pressure related to carcinogen-induced unscheduled DNA synthesis, DNA carcinogen binding, and chromosomal aberrations in human lymphocytes.

Unscheduled DNA synthesis (excision-repair) of N-acetoxy-2-acetylaminofluorene (NA-AAF) damage to the DNA of human lymphocytes was determined quantitatively for 92 individuals with diastolic blood pressures ranging from 65 to 120 mm of Hg (8,7 to 16 kPa). Measurements of NA-AAF-induced repair synthesis (incorporation of[3H]thymidine in the presence of 10 mM hydroxyurea) showed linear increase with the blood pressure in the individuals under study. Concurrent determinations for the levels of 3H-labeled 7,12-dimethyl-benz[a]anthracene bound to the DNAs of lymphocytes after 18 hr of culturing have shown that increased amounts of DNA bound carcinogen were linearly proportional to increased NA-AAF-induced repair synthesis values, and therefore were correlated to high blood pressure. The number of NA-AAF-induced chromosomal abberations in lymphocytes increased linearly with the diastolic blood pressures of the individuals. High NA-AAF-induced repair synthesis values also tended to be associated with increased NA-AAF-induced chromosomal damage. Together, these results suggest that individuals with elevated blood pressures have a greater potential for accumulating DNA damage, because of an increased chemical reactivity of lymphocytes to carcinogen exposure, than do individuals with normal blood pressure.

Hybridization of polyuridylic acid to human DNA immobilized onto nitrocellulose filters.

The level of deoxyadenylate (da) regions in human DNA was estimated from formation of poly(U)-poly(da) triplexes on nitrocellulose filters that were RNAase resistant. The (dA) rich sequences were determined following mild ribonuclease treatment of the poly(U)-DNA hybrids (5 mug/ml at 25 degreesC for 30 min), where as exhaustive ribonuclease treatment (5 mug/ml at 25 degrees C for 6 hr) estimated the more (dA) pure sequences. The level of (dA) rich regions was 0.39% of the DNA and for the more (dA) pure regions it was 0.07%. The (dA) regions were widely distributed throughout human DNA regardless of base composition or sequence repetition. However, a concentration of (dA) regions into main band CsC1 gradient fractions of DNA and into repeated DNA was observed.