Pain syndromes in HIV infection.

Pain causes considerable disability and discomfort in HIV (Human Immunodeficiency Virus) infected individuals. A large number of patients infected with HIV suffer from one or more pain-related syndromes. Pain is under-reported and suboptimally managed in these patients. An outline of the different pain syndromes, including headache, oral cavity pain, chest pain, abdominal pain, anorectal pain, musculoskeletal pain and peripheral neuropathic pain, and their aetiologies are discussed. Current pain management modalities, including non-narcotic and narcotic analgesics, tricyclic antidepressants, anticonvulsants, physical therapy and psychological techniques, are outlined. Treatment should be based on the same principles applied to the management of cancer-related pain. A multi-disciplinary, comprehensive approach to pain management will assist these individuals to achieve improved levels of comfort, function and quality of life in this ultimately terminal illness.


Health and the environment: a global challenge. WHO Commission on Health and Environment.

A healthy environment is not only a need, it is also a right; the right to live and work in an environment conducive to physical and mental health is enshrined in the Universal Declaration of Human Rights. Everyone shares the responsibility for ensuring that this right is duly acknowledged. The responsibility for action lies with individuals and with business. Governments have the responsibility of setting up the strategic and institutional framework within which action is taken. There are three main global objectives: achieving a sustainable basis for health for all--by slowing down population growth as soon as possible, and promoting life-styles and patterns of consumption among affluent groups and countries that are consistent with ecological sustainability; providing an environment that promotes health--by reducing the risk of physical, chemical and biological hazards and ensuring that everyone has the means to acquire the resources on which health depends; making all individuals and organizations aware of their responsibilities for health and its environmental basis.


A comparison of group A streptococcal serotypes isolated from the upper respiratory tract in the USA and Thailand: implications.

Characterization of group A beta-haemolytic streptococci in upper respiratory tract isolates from the USA and Thailand revealed that whereas 80% of the U.S. isolates could be M or opacity factor (OF) typed, less than 20% of the Thai isolates could be characterized with the available typing sera (P less than 0.001). There was also a statistically significant difference observed in the percentage of strains that could be characterized by the T-agglutination pattern (93% in the USA vs 61% in Thailand, P less than 0.001). Even among the identifiable strains, marked differences in the distribution of the recovered serotypes were noted between the two countries. These results show that there are a significant number of as yet unidentified group A streptococcal strains in parts of the world where streptococcal infections and their sequelae are important public health problems. They further imply that such findings must be taken into consideration in the future when designing possible streptococcal vaccines for worldwide use.


Level of serum antibodies to mycobacterial antigens in healthy Czechs.

Levels of IgG antibodies to Mycobacterium bovis BCG and M. avium antigens were examined by enzyme-linked immunosorbent assay (ELISA) in samples of sera from 898 healthy adults aged greater than 40 years and 1170 children aged 1-6 years, selected at random in 10 districts of Czechoslovakia. The median antibody titre to M. bovis BCG in adults ranged from 1:28 to 1:161.7 and that in children from 0 to 1:50. The mean titres of M. avium antigen ranged from 1:2 to 1:21 in adults and from 0 to 1:8 in children.


Rabies in China: recommendations for control.

Reviewed are the results of 15 years' experience with rabies at You-An Infectious Disease Hospital, Beijing, China. The purpose of the study was to determine whether there are any epidemiological or clinical features of rabies that are unique to China and which might be important in developing a strategy to control it. During the period under study, 64 patients with rabies were admitted to You-An Hospital. Exposure to dogs was associated with 61 cases, two involving the handling of dog carcasses that were being prepared for meals. All of the exposures occurred in rural areas, and none of the patients received adequate prophylaxis. Patients with proximal sites of exposure and with severe injuries developed rabies after short incubation periods (P less than 0.05, and P less than 0.02, respectively). Failed vaccination was also associated with a short incubation period (P less than 0.05). Haematemesis occurred in 20 patients and was associated with shorter incubation periods (P less than 0.02), facial exposure sites (P = 0.021), and severe injuries (P = 0.047). A strategy to control rabies in China should include efforts to educate the public about handling the carcasses of stray dogs, in addition to the currently recommended strategy of controlling the dog population and of vaccinating domesticated animals.


[Trachoma in the province of Ouarzazate, Morocco].

A survey on the prevalence and severity of trachoma was carried out in the province of Ouarzazate, Morocco. In conformity with the guidelines proposed by the WHO Programme for the Prevention of Blindness, a random sample of 30 clusters was extracted from the general population of the province, according to probability proportional to size. Thus, the sample comprised 1200 individuals, of whom 1185 were examined. Participation in the survey was 98.8% and, overall, the sample is considered representative of the province. The simplified grading system proposed by WHO was used to register the data on trachoma and its complications. The global prevalence of trachoma was estimated at 40.8% (95% confidence interval (95% CI) = 30.2-51.4%) and that of active trachoma (follicular (TF), intense (TI), and mixed (TF + TI)) at 18% (95% CI = 12.8-23.2%). The trachomatous intensity indicator (presence of TI) for children under 10 years of age was 12.8% (95% CI = 6.8-18.8%). The severity of the infection is confirmed by prevalences of trichiasis-entropion of 2.2% (95% CI = 1.4-3.0%) and central corneal opacity of 3.3%. Corneal blindness is estimated at 1.6%. The epidemiological pattern of trachoma merits particular attention in the field of public health, particularly in the valley of Oued Drâa, where all the indicators are consistently higher than those elsewhere in the province.


Low seroconversion rates to measles vaccine among children in Nigeria.

The Nigerian Expanded Programme on Immunization (EPI) was assessed with particular reference to measles immunization. Of 150 children who received measles vaccine at the Institute of Child Health, University of Ibadan, Nigeria, 82 (54.7%) seroconverted. The immune response was directly related to the titre of the vaccines used. Vaccines whose titres were 10(-1) to 10(1.7) stimulated immune responses in 0-25% of vaccinees, those with titres in the range 10(-2.1) to 10(-2.5) stimulated responses in 12-47.6%, while those with titres of 10(-2.7) to 10(-3.4) stimulated responses in 87.5-100% of vaccinees. Only one of the vaccines used had a titre that met the minimum WHO required standard of log 10(-3) TCID50 at the point of vaccination.


Immunization coverage in India for areas served by the Integrated Child Development Services programme. The Integrated Child Development Services Consultants.

The Integrated Child Development Services (ICDS) programme was launched by the Indian government in October 1975 to provide a package of health, nutrition and informal educational services to mothers and children. In 1988 we studied the impact of ICDS on the immunization coverage of children aged 12-24 months and of mothers of infants in 19 rural, 8 tribal, and 9 urban ICDS projects that had been operational for more than 5 years. Complete coverage with BCG, diphtheria-pertussis-tetanus (DPT) and poliomyelitis vaccines was recorded for 65%, 63%, and 64% of children, respectively, in the ICDS population. By comparison, the coverage in the non-ICDS group was only 22% for BCG, 28% for DPT, and 27% for poliomyelitis. Complete immunization with tetanus toxoid was recorded for 68% of the mothers in the ICDS group and for 40% in the non-ICDS group. Coverage was greater in the urban and lower in the tribal projects. Scheduled castes, scheduled tribes, backward communities, and minorities (groups that have a high priority for social services) had immunization coverages in ICDS projects that were similar to those of higher castes.


Breast-feeding, nutritional status, and other prognostic factors for dehydration among young children with diarrhoea in Brazil.

Early identification of children at high risk of diarrhoea-associated dehydration would be of great value to health care workers in developing countries. To identify prognostic factors for life-threatening dehydration, we carried out a case-control study among under-2-year-olds in Porto Alegre, Brazil. Cases were 192 children admitted to hospital with moderate or severe dehydration, while controls were children matched to controls by neighbourhood and age, who experienced nondehydrating diarrhoea in the week preceding the interview. The following variables were significantly associated with an increased risk of dehydration, after adjustment for age and other confounding variables: absence of the father from the home; low paternal education level; young age; maternal age 25-29 years or less than 20 years; mother of mixed race; high birth order; short birth interval; low birth weight; stunting, underweight and wasting; lack of breast-feeding; presence of other under-5-year-olds in the home; families with 4-5 members; lack of antenatal care; less than three doses of diphtheria-pertussis-tetanus or poliomyelitis vaccine; previous admission to hospital; use of medicines during the fortnight prior to the episode; and living in an unclean home. The associations were particularly strong (P less than 0.001) for the child's age, birth weight and other anthropometric indicators, birth interval, and feeding mode. In terms of their sensitivity and specificity, however, these prognostic factors were not as effective as early signs and symptoms for predicting the outcome of the episode.


Evaluating the efficacy of chloroquine in febrile Guinean children infected with Plasmodium falciparum by a simplified in vivo test.

A modified version of a simplified in vivo test was applied to assess the susceptibility of Plasmodium falciparum to chloroquine among semi-immune febrile children in Kouroussa, Guinea. In 27% of cases, a partial response (RI/RII) to chloroquine was observed; the remainder showed a good response (S/RI). This test is both practical and useful to assess the efficacy of chloroquine at the intermediate level of health care services.


Environmental pollution and chronic arsenicosis in south Calcutta.

Careless handling of industrial wastes often creates problems for human health and the environment. Chronic arsenic toxicity, resulting from household use of arsenic-contaminated water occurred in 53 out of 79 members (67%) of 17 families residing in South Calcutta close to a factory that manufactured Paris-green (copper acetoarsenite). Clinical investigation of 20 of these affected persons showed typical skin pigmentation as well as palmar and plantar keratosis in all of them, while gastrointestinal symptoms, anaemia and signs of liver disease and peripheral neuropathy were seen in many. The water used by the affected families for drinking and cooking had been taken from shallow tubewells and had arsenic levels from 5.0 to 58 mg/l (WHO permissible limit, 0.05 mg/l). Other residents in the same area whose drinking-water came from deep tubewells or from tap water supplied by the Calcutta Municipal Corporation (arsenic levels, less than 0.05 mg/l) were not affected. The study confirms that arsenic in the shallow tubewells was due to the waste discharged by the factory producing Paris-green.


Epidemiology's contribution to health service management and planning in developing countries: a missing link.

Two hypotheses are examined in the light of experience and the literature: (1) health service planning requires little epidemiological information, and (2) health services rarely get useful answers to relevant epidemiological questions. In the first hypothesis, the theoretical robustness of the concept of a minimum package of activities common to all facilities belonging to the same level of the system and the extent to which it is unaffected by variations in the frequencies of most diseases are examined. Semi-quantitative analyses and analysis of routine entries and participation suffice to adapt this package to the local context. Some of the methods which give a fundamental role to epidemiological information are criticized. With regard to the second hypothesis, the pertinent contributions epidemiology may make to health service organization are reviewed. These include identification of diseases that justify special activities (health maps and interepidemic surveillance), determination of the activities that should be added to the health centres, the political usefulness of rare impact assessments, and the relevant demographic elements. Finally an epidemiological agenda is proposed for specialized centres, districts, universities, and the central decision-making level of health ministries in developing countries.


Diagnosis of causes of childhood deaths in developing countries by verbal autopsy: suggested criteria. The SEARCH Team.

In the absence of medical certification of deaths in developing countries, lay reporting and verbal autopsy have emerged as useful alternative methods for collecting data on causes of death. Of these, verbal autopsy offers advantages and is widely used in field studies and child survival programmes. However, because uniform and valid criteria for the diagnosis of common causes of death are lacking, comparison of the results of different studies becomes meaningless. This article proposes such a set of criteria for the cause of death among neonates and for those aged 1-59 months. The criteria are based on the findings of earlier validation studies, a Delphi survey and the experience gained from performing 1000 verbal autopsies in Gadchiroli, India. The emergence of such standardized criteria of causes of death should be of immense value for health planning, monitoring and evaluation purposes and for interregional comparisons.


Risk factors for injuries due to the 1990 earthquake in Luzon, Philippines.

On 16 July 1990, an earthquake measuring 7.7 on the Richter scale struck the island of Luzon, Philippines. A case-control study was carried out to identify the risk factors for earthquake-related injuries and at the same time observations were made on the rescue efforts. Being hit by falling objects was the leading cause of injury (34%). Those injured during the tremor were more likely to have been inside buildings constructed of concrete or mixed materials (odds ratio, 2.6; 95% confidence interval (CI), 1.7-4.1) and to have been on the middle floors of multistorey buildings (odds ratio, 3.4; 95% CI, 2.2-5.5). Leaving a building during the earthquake was a protective behaviour (odds ratio, 0.3; 95% CI, 0.2-0.8). Of the 235 survivors who were trapped and rescued alive from the rubble, 99% were rescued within 48 hours of the impact of the tremor. These findings should prove useful in developing seismic safety codes. People should be taught proper evasive actions to take during earthquakes, and training in basic first aid and methods of rescue should be an integral part of community preparedness programmes.


HIV-associated tuberculosis in developing countries: clinical features, diagnosis, and treatment.

This article reviews the clinical aspects and diagnosis of HIV-associated tuberculosis in developing countries, and summarizes WHO's recommendations for treatment. According to WHO estimates (early 1992) over 4 million persons worldwide have been infected with HIV and tuberculosis; 95% of them are in the developing countries. Clinical features of HIV-associated pulmonary tuberculosis in adults are frequently atypical, particularly in the late stage of HIV infection, with non-cavitary disease, lower lobe infiltrates, hilar lymphadenopathy and pleural effusion. More typical post-primary tuberculosis with upper lobe infiltrates and cavitations is seen in the earlier stages of HIV infection. Extrapulmonary tuberculosis is reported more frequently, despite the difficulties in diagnosing it. WHO's recent guidelines recommend 6-month short-course chemotherapy with isoniazid, rifampicin, pyrazinamide and ethambutol for patients with HIV-associated tuberculosis. The older 12-month regimen without rifampicin is much less effective. Streptomycin should not be used, because of the risk of transmitting blood-borne pathogens through contaminated needles. Thioacetazone should be abandoned, because of severe adverse reactions observed among HIV-infected patients. The roles of preventive chemotherapy and BCG vaccination for prevention of tuberculosis are also briefly discussed.


Nomenclature for human complement component C2. WHO-IUIS Nomenclature Sub-Committee.

This note describes the designations for variants of the human complement component C2, which were approved by the Nomenclature Committee of the International Union of Immunological Societies (IUIS).


Revised nomenclature for human complement component C4. WHO-IUIS Nomenclature Sub-Committee.

This note describes the recommended designations for allotypes of human complement component C4, which were approved by the Nomenclature Committee of the International Union of Immunological Societies (IUIS).


Nomenclature for human complement factor B. WHO-IUIS Nomenclature Sub-Committee.

In this note is recommended a unified nomenclature for allotypes and variants of human complement factor B, which was approved by the Nomenclature Committee of the International Union of Immunological Societies (IUIS).


Identification of genes involved in programmed cell death.

Three modes of activation of apoptosis are described: induction, in which new gene expression occurs after the stimulus is applied; transduction, in which gene expression is unnecessary at the time of stimulation; and release, in which apoptosis is activated by the inhibition of gene expression. Genes activated in the induction mechanism were identified by a process of subtractive hybridization, whereby newly transcribed messenger RNAs could be isolated. Progress in characterizing some of these genes is described. There are many difficulties and conceptual problems associated with such a gene cloning approach, but the results will be worth the effort.


The significance of spontaneous and induced apoptosis in the gastrointestinal tract of mice.

The crypts of the gastrointestinal mucosa are highly structured and polarised organs with rapid cell proliferation and an hierarchical organisation with relatively few stem cells. These tend to be located at specific positions in the tissue--at the crypt base in the colon and about four cell positions from the base (above the Paneth cells) in the small intestine. A small but constant level of spontaneous cell death occurs in the crypt. The levels of cell death are elevated by small exposures to radiation or cytotoxic drugs. The morphology of the cell death is typical of apoptosis. The maximum yield of cell death following cytotoxic exposure is observed at about 3-6 h after treatment and for many agents the death is characteristically located at the fourth (stem) cell position in the small intestine. The significance and implications of these observations are discussed in relation to the internal screening and programming within damage cells and with respect to tissue homeostatic mechanisms.


Apoptosis in the development of the immune system: growth factors, clonal selection and bcl-2.

The mammalian immune system is essential for surviving challenge infections with a great range of potential pathogens. The protective effect produced is dependent on many different types of cells which require flexible and independent production and regulation. In particular, many important responses are carried out by lymphocytes, which recognise foreign antigen through exquisitely specific receptors: i.e. surface immunoglobulin (sIg) on B lymphocytes and the T cell receptor (TCR) on T lymphocytes. Each lymphocyte displays receptors with a single specificity, allowing cells with particular specificities to be regulated independently. Since millions of different Igs and TCRs are expressed, the precise selection and regulation of each T and B cell population to produce a useful self-tolerant repertoire is a very complex process. Control of cell populations can, in theory, be exercised at a number of levels, including modulation of active cell death by apoptosis. Recent research has demonstrated that regulation of apoptosis is indeed a crucial element in the control of the immune system in general, and in the development of the TCR and Ig repertoires in particular. The molecular analysis of apoptosis now takes a high priority and the proto-oncogene bcl-2 appears to be responsible for specific suppression of apoptosis in several important situations. It is also clear that malfunctions affecting apoptosis, and in particular bcl-2, can result in significant progression towards malignancy.


Factors that influence the therapeutic activity of 5-fluorouracil [6RS]leucovorin combinations in colon adenocarcinoma xenografts.

The therapeutic activity of FUra alone or combined with [6RS]LV doses ranging from 50 to 1,000 mg/m2 was examined in eight colon adenocarcinoma xenografts, of which five were established from adult neoplasms (HxELC2, HxGC3, HxVRC5, HxHC1, and HxGC3/c1TK-c3 selected for TK deficiency) and three were derived from adolescent tumors (HxSJC3A, HxSJC3B, and HxSJC2). The growth-inhibitory effects of FUra were potentiated by higher doses of [6RS]LV (500-1,000 mg/m2) in three lines (HxGC3/c1TK-c3, HxSJC3A, and HxSJC3B) and by a low dose of [6RS]LV in only one tumor (HxVRC5). Expansion of pools of CH2-H4PteGlun+H4PteGlun (greater than or equal to 2.4-fold) in response to higher doses of [6RS]LV was obtained in all lines except HxHC1. Metabolism of [6RS]LV was high in HxVRC5, with high levels of 5-CH3-H4PteGlu being detected, but not in HxHC1, in which levels of 5-CH3-H4PteGlu and CH = H4PteGlu+10-CHO-H4PteGlu remained relatively low. In the adolescent tumors, levels of CH = H4PteGlu+10-CHO-H4PteGlu were consistently higher than those of 5-CH3-H4PteGlu following [6RS]LV administration, and in HxSJC3A, in which pools of CH2-H4PteGlun+H4PteGlun were significantly expanded, 5-CH3-H4PteGlu concentrations were lower than those observed in the other two lines. The sensitivity of tumors to FUra +/- [6RS]LV and the characteristics of [6S]LV metabolism did not correlate with the activity of CH = H4PteGlu synthetase, the enzyme responsible for the initial cellular metabolism of [6S]LV to CH = H4PteGlu. Thus, no single metabolic phenotype correlated with the [6RS]LV-induced expansion of CH2-H4PteGlun+H4PteGlun pools. Potentiation of the therapeutic efficacy of FUra by [6RS]LV was observed in HxGC3/c1TK-c3 xenografts but not in parent HxGC3 tumors, demonstrating the influence of dThd salvage capability in the response to FUra-[6RS]LV combinations. Plasma dThd concentrations in CBA/CaJ mice were high (1.1 microM). The present data therefore demonstrate the importance of (1) higher doses of [6RS]LV, (2) expansion of pools of CH2-H4PteGlun+H4PteGlun, and (3) dThd salvage capability in potentiation of the therapeutic efficacy of FUra in colon adenocarcinoma xenografts. The plasma levels of FUra achieved in mice are presented.


Influence of the cardioprotective agent dexrazoxane on doxorubicin pharmacokinetics in the dog.

The influence of dexrazoxane on doxorubicin pharmacokinetics was investigated in four dogs using the two treatment sequences of saline/doxorubicin or dexrazoxane/doxorubicin. Intravenous doses of 1.5 mg/kg doxorubicin and 30 mg/kg (the 20-fold multiple) dexrazoxane were given separately, with doxorubicin being injected within 1 min of the dexrazoxane dose. Both doxorubicin and its 13-dihydro metabolite doxorubicinol were quantified in plasma and urine using a validated high-performance liquid chromatographic (HPLC) fluorescence assay. The doxorubicin plasma concentration versus time data were adequately fit by a three-compartment model. The mean half-lives calculated for the fast and slow distributive and terminal elimination phases in the saline/doxorubicin group were 3.0 +/- 0.5 and 32.2 +/- 12.8 min and 30.0 +/- 4.0 h, respectively. The model-predicted plasma concentrations were virtually identical for the saline and dexrazoxane treatment groups. Analysis of variance of the area under the plasma concentration-time curve (AUCo-infinity), terminal elimination rate (lambda z), systemic clearance (CLs), and renal clearance (CLr) for the parent drug showed no statistically significant difference (P greater than 0.05) between the two treatments. Furthermore, the doxorubicinol plasma AUCo-t value and the doxorubicinol-to-doxorubicin AUCo-t ratio showed no significant difference, demonstrating that dexrazoxane had no effect on the metabolic capacity for formation of the 13-dihydro metabolite. The total urinary excretion measured as parent drug plus doxorubicinol and the metabolite-to-parent ratio in urine were also unaffected by the presence of dexrazoxane. The myelosuppressive effects of doxorubicin as determined by WBC monitoring revealed no apparent difference between the two treatments. In conclusion, these results show that drug exposure was similar for the two treatment arms. No kinetic interaction with dexrazoxane suggests that its coadministration is unlikely to modify the safety and/or efficacy of doxorubicin.


Effect of coadministration of selenite on the toxicity and antitumor activity of cis-diamminedichloroplatinum (II) given repeatedly to mice.

The effect of selenite coadministration on the toxicity and antitumor activity of repeated treatment with high doses of cis-diamminedichloroplatinum (cis-DDP) was examined in mice. Sodium selenite was injected s.c. into separate abdominal sites of mice together with cis-DDP at a molar ratio of 1:3.5 (selenite to cis-DDP) on day 0. The same amount of selenite was given daily for 4 subsequent days (days 1-4). This fixed administration schedule was repeated weekly for a total of 7 weeks. Under the experimental conditions used, the lethal toxicity, renal toxicity [indicated by an increase in blood urea nitrogen (BUN) and plasma creatinine levels], hepatic toxicity (indicated by an increase in plasma GPT and GOT activity), and myelotoxicity (indicated by a decrease in the numbers of leukocytes and platelets) observed in mice given repeated doses of cis-DDP alone (15 or 25 mumol/kg, s.c.) were significantly depressed by the coadministration of sodium selenite. Treatment with cis-DDP alone (15, 20, or 25 mumol/kg, s.c.) resulted in some dose-dependent prolongation of the life span of mice transplanted either s.c. with colon adenocarcinoma 38 (colon 38) or i.p. with P388 leukemia (P388) but did not completely depress the tumor growth, and the animals died of either progressive disease or cis-DDP-induced toxicity. However, following the coadministration of 7.1 mumol/kg selenite with 25 mumol/kg cis-DDP, all of the mice transplanted either s.c. with colon 38 or i.p. with P388 survived for as long as 4 months after the end of the treatment and showed no evidence of malignancy. These results indicate that selenite coadministration enables the use of increasing doses of cis-DDP and, consequently, enhances the antitumor effect of cis-DDP by depressing its side effects.


Lack of involvement of reactive oxygen in the cytotoxicity of mitoxantrone, CI941 and ametantrone in MCF-7 cells: comparison with doxorubicin.

The MCF-7 cell S9 fraction and whole MCF-7 cells can mediate one-electron-redox cycling of doxorubicin, giving rise to concomitant oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH), formation of a drug semiquinone free radical, consumption of molecular oxygen and formation of superoxide anions and hydroxyl radicals. Doxorubicin redox cycling was consistent with DNA strand breakage and cell kill in MCF-7 cells. In contrast, no evidence for redox cycling was found for mitoxantrone (MIT), CI941 or ametantrone (AMET) in MCF-7 cells. Despite the absence of redox cycling, the CI941, MIT, and AMET concentrations resulting in 50% mortality (LC50; 1.5 x 10(-10), 5.2 x 10(-9) and 1.2 x 10(-6) M, respectively) of MCF-7 cells were lower than that of DOX (3.0 x 10(-6) M). Furthermore, the higher cytotoxicity of MIT and CI941 as compared with AMET or DOX was associated with greater efficiency in inducing DNA strand breakage in MCF-7 cells as determined by alkaline elution. Since MIT and CI941 proved to be the most potent DNA-damaging and cytotoxic agents in this study, the ability of DOX to undergo redox cycling does not appear to confer increased cytotoxic potential on this agent. The present study revealed several important aspects with regards to the structural modification of anthraquinone antitumour agents. Firstly, the C1 and C4 positioning of the hydroxyethylamino side chains on MIT, CI941 and AMET is associated with a lack of flavin reductase-mediated activation of these agents. Secondly, the possession of a C5 or C8 aromatic hydroxyl group appears to be intimately involved in the enhanced DNA strand breakage and cytotoxic potency of MIT and CI941, since AMET does not possess these groups. These findings indicate that future development of quinone antitumour agents should concentrate on compounds that do not undergo redox cycling but do possess aromatic hydroxyl groups, since the latter appear to be responsible for the enhanced cytotoxicity of MIT and CI941.


Activity of aphidicolin glycinate alone or in combination with cisplatin in a murine ovarian tumor resistant to cisplatin.

Aphidicolin, a reversible inhibitor of DNA polymerase alpha and delta, has recently been reported to reverse the resistance to cisplatin (DDP) of an ovarian cancer cell line. We investigated the pharmacokinetics of aphidicolin in mice and examined its activity either alone or in combination with DDP in the DDP-sensitive M5076 (M5) murine reticular cell sarcoma as well as in a DDP-resistant subline (M5/DDP). The drug was cleared from plasma very rapidly (clearance, 41.6 ml min-1 kg-1), showing a half-life of 15 min. Aphidicolin concentrations in the tumor were approximately 50% of those found in plasma at steady state. Using several dose schedules and continuous infusions we failed to detect significant antitumor activity for aphidicolin glycinate. Potentiation of the activity of DDP by aphidicolin glycinate was moderate in mice bearing M5 tumor as well as in those bearing M5/DDP tumor. These data do not support the possible clinical use of aphidicolin in combination with DDP. However, further studies should be carried out in different tumor models before this possibility is conclusively ruled out.


Drug resistance as a dynamic process in a model for multistep gene amplification under various levels of selection stringency.

Resistance to antineoplastic drugs has been a major impediment to the successful treatment of cancer. Recent studies suggest that several mechanisms are responsible for the emergence of drug resistance but that high levels of resistance and poor prognosis are strongly associated with gene or oncogene amplification. In this report we describe a probabilistic model for gene amplification in a tumor that grows under various drug protocols. The model is new in that it treats drug resistance as a dynamic process and examines specific assumptions about the underlying molecular events. Using this model, we specify the conditions for the emergence of drug-resistant mutants prior to selection as well as the relationship between the stringency of the selecting environment and the characteristics of the resultant cellular phenotype.


Adjuvant chemotherapy with vinblastine, adriamycin, and UFT for renal-cell carcinoma.

VAU therapy (vinblastine, Adriamycin, and UFT) was given postoperatively to 31 patients with stage I, II, or III renal-cell carcinoma, and the incidence of relapse as well as the survival of patients were studied. Administration was started at 7-14 days post-surgery; 5 mg/m2 vinblastine and 30 mg/m2 Adriamycin were given i.v. once every 4 weeks for a total of five courses, and three capsules of UFT (containing 300 mg tegafur) were given p.o. every day for 2-3 years. The postoperative observation period ranged from 2 years and 6 months to 7 years and 1 month (mean, 4 years and 2 months). The 1-year survival of patients was 100%, and the 3- and 5-year survival values were 96%. These results were significantly better (P less than 0.01) than the respective values (81%, 72%, and 60%) obtained for the historical controls, i.e., the 60 patients with stage I, II, or III renal-cell carcinoma who received no chemotherapy. Side effects such as alopecia, gastrointestinal symptoms, and myelosuppression were encountered, but all symptoms were so mild and transient that discontinuation of the treatment was not necessary. As VAU therapy might be useful as adjuvant chemotherapy for renal-cell carcinoma, it seems to merit further study.


A preliminary clinical study of gossypol in advanced human cancer.

A total of 34 patients with advanced cancer were given weekly or daily escalating doses of oral gossypol, a cottonseed-oil constituent showing evidence of antineoplastic activity in pre-clinical studies. No major adverse events occurred and there was no evidence of haematological or biochemical disturbance. As determined by dose escalation in 17 patients, the dose-limiting toxicity was emesis in 16 patients. There was no evidence of tumour regression in any of the 20 patients assessed for response. We conclude that gossypol is safe but unlikely to be clinically useful in patients with advanced cancer.


Inhibition of growth factor binding and intracellular Ca2+ signalling by dextran sulfates of different sizes and degrees of sulfation.

The ability of dextran sulfates of varying molecular sizes (5-500 kDa) and degrees of sulfate substitution (0.3-1.9) to inhibit the binding of platelet-derived growth factor (PDGF) to intact Swiss 3T3 fibroblasts and to inhibit inositol(1,4,5)trisphosphate-dependent release of Ca2+ in permeabilized Swiss 3T3 cells was examined in the present study. Significant correlations were found between increased molecular size of the dextran sulfates and inhibition of both PDGF binding (r = 0.77) and Ca2+ release (r = 0.72). The degree of sulfate substitution did not correlate with inhibition of either activity.


In vivo evidence of complete circumvention of vincristine resistance by a new triazinoaminopiperidine derivative S 9788 in P388/VCR leukemia model.

S 9788, a new triazinoaminopiperidine derivative, was found to be a potent reversant of vincristine resistance in the in vivo murine leukemic P388/VCR model. In two treatment regimens (Q4D days 1, 5 and 9 and QD days 1-9), S 9788 enhanced the antitumor activity of vincristine in a dose-dependent manner, resulting in a complete circumvention of drug resistance for well-tolerated doses of S 9788. S 9788 was also effective in enhancing therapeutic effects of vincristine in the treatment of sensitive P388-bearing mice. These results strongly suggest that S 9788 may be a potential candidate for circumvention of multidrug resistance (MDR) in clinical practice.


Intra-arterial administration of methotrexate, adriamycin, and cisplatin as neoadjuvant chemotherapy for bladder cancer.

As neoadjuvant chemotherapy for advanced bladder cancer, the intra-arterial administration of methotrexate (MTX), Adriamycin (ADM), and cisplatin (CDDP; IA-MAC) was evaluated. A total of 48 patients with bladder cancer (greater than or equal to T2 or CIS) were selected and received 30.1 mg MTX, 34.5 mg ADM, and 89.1 mg CDDP as an average course. The mean tumor-regression rate after 2 or 3 weeks was 52.3%, and patients with grade 3 transitional-cell carcinoma showed the best results, achieving a 69.6% regression rate. In 30 cases (63%), downstaging was observed. Among the 46 patients who underwent subsequent surgical therapy, the bladder could be preserved in 26 cases by transurethral resection or segmental resection. According to the criteria of the Japanese Association of Cancer Therapy, a histological effect of GIII or better was obtained in 15 cases (29%). The histological effect correlated well with the tumor-regression rate. As compared with intravenous therapy with MTX, vinblastine, ADM, and CDDP (M-VAC), IA-MAC treatment was well tolerated due to its lower degree of bone marrow suppression, and it resulted in a longer disease-free interval and better survival. In addition, the period prior to surgical therapy was shortened in this study. These results suggest that IA-MAC chemotherapy can be useful as an arm of multidisciplinary treatment of advanced bladder tumors.


The 4th study of prophylactic intravesical chemotherapy with adriamycin in the treatment of superficial bladder cancer: the experience of the Japanese Urological Cancer Research Group for Adriamycin.

A multicentric randomised trial was conducted for the purpose of investigating the efficacy of intravesical chemoprophylaxis of superficial bladder cancers. A total of 443 patients (number of evaluable patients, 284) were registered from July 1987 to December 1989 and randomised into 3 groups. Group A received 21 intravesical instillations of Adriamycin (ADM) at 20 mg/40 ml physiological saline for 2 years after undergoing transurethral resection (TUR); group B was given the same dose as group A but received 6 intravesical instillations for 2 weeks before undergoing TUR; and group C served as a control and underwent TUR only. Better prophylactic effects were obtained in group A. The overall non-recurrence rates calculated for groups A and B differed significantly (P less than 0.05) on day 240, and those determined for groups A and C were also significantly different (P less than 0.01) on day 480. No benefit was obtained using intravesical instillation prior to TUR (group B). The major side effects encountered were pollakisuria and miction pain, which occurred in 32% of the patients in group A and in 52% of those in group B.


Long-term results of intravesical chemoprophylaxis of superficial bladder cancer: experience of the Japanese Urological Cancer Research Group for Adriamycin.

Long-term results were analyzed in terms of tumor progression and survival in patients with superficial bladder cancer who were enrolled in the second intravesical chemoprophylactic study of the Japanese Urological Cancer Research Group for Adriamycin, which was started in July 1982. This study was a prospective, randomized, controlled trial conducted on primary tumors treated with a long-term instillation regimen that involved control versus intravesical instillations of Adriamycin or mitomycin C given once a week for the first 2 weeks, once every other week for 14 weeks, once a month for 8 months, and once every 3 months for 1 year, for a total of 21 instillations in 2 years. An analysis of the prophylactic effects of such treatment on bladder tumors after TUR has previously been performed, and the results have been published elsewhere. The present study represents a follow-up of the above trial. Of the 671 cases previously analyzed with regard to tumor prophylaxis, 158 cases (23.5%) were eligible to be followed for tumor progression and survival. A detailed comparison of the background factors between these 158 patients and the other 513 cases revealed no statistically significant difference. Thus, the 158 evaluable cases might reasonably be considered to represent all patients enrolled in the second study, and the results were thought to be reasonable enough to reflect the long-term efficacy of the long-term instillation regimen adopted in this study. The median follow-up for these 158 cases was 6.6 years. Tumor progression in terms of the disease stage and/or grade occurred in 43 of 127 patients who received prophylactic instillations and in 12 of 31 control cases. No significant difference in the incidence of tumor progression was found between the treatment and the control groups. In addition, no difference in survival was observed between the treatment group and the control group. Survival was also compared between patients who showed tumor progression and those who did not. All patients whose tumors did not progress survived, whereas the 7-year survival of those exhibiting tumor progression was less than 90%.


Prophylactic chemotherapy for primary and recurrent superficial bladder cancer: preliminary results. The Hokkaido University Bladder Cancer Collaborating Group.

A multicenter trial for postoperative prophylaxis of the recurrence of superficial Ta-T1, G1-G2 bladder cancer was performed. Eligible patients with primary or recurrent superficial bladder cancer were randomized into four groups. For the primary cases, intravesical instillation of drugs [group A, 20 mg Adriamycin (ADM) + 200 mg cytosine arabinoside (CA) in 30 ml physiological saline; group B, 10 mg peplomycin (PEP) + 200 mg CA in 30 ml physiological saline; group C, 2 mg neocarzinostatin (NCS) + 200 mg CA in 30 ml physiological saline; and group D, control] was carried out once a week for 2 weeks, once every 2 weeks for 14 weeks, once monthly for 8 months, and, finally, once every 3 months for 1 year. For the recurrent cases, intravesical instillation of 20 mg ADM + 200 mg CA in 30 ml physiological saline as described above and daily oral administration of another drug [group E, 300 mg/day UFT; group F, 200 mg 5-fluorouracil (5-FU)/day; group G, 30 mg ubenimex/day; and group H, no oral drug] was performed. The postoperative follow-up period was 3-36 months. A total of 193 primary cases and 121 recurrent cases of superficial bladder cancer were evaluated. The cumulative 12-month nonrecurrence rates for the primary cases were 86.2% in group A, 78.1% in group B, 82.1% in group C, and 68.4% in group D. The cumulative nonrecurrence rate obtained using ADM+CA (group A) was significantly higher than the control value. On the other hand, no significant difference was found in the cumulative nonrecurrence rates calculated for the recurrent cases, regardless of the oral drug given. Intravesical instillation of ADM+CA for primary superficial bladder cancer was considered to be useful, but the long-term effect of intravesical instillation remains to be elucidated. Further refinement of this regimen is necessary for effective prophylaxis of the recurrence of superficial bladder cancer.


A prospective randomized study of prophylaxis of tumor recurrence following transurethral resection of superficial bladder cancer--intravesical thio-TEPA versus oral UFT.

The long-term prophylactic effect of chemotherapy following transurethral electroresection of bladder tumors (TUR-Bt) was investigated using three different modalities: no prophylactic treatment (group C); oral UFT given at 1296 mg/day for 2 years (group U); and intravesical thio-TEPA at 30 mg/30 ml physiological saline, instilled 32 times over 2 years (group T). Patients newly diagnosed as having superficial bladder cancer (stage, less than or equal to pT1b; grade, less than or equal to G2) who had undergone TUR-Bt at Nara Medical University and its affiliated hospitals between November 1986 and March 1990 were allocated to one of the three groups by the envelope method. The initial treatment was maintained until the third recurrence or disease progression, except for TUR-Bt which was performed at the time of recurrence. The registered cases included 51 patients in group C, 50 in group U, and 52 in group T, and the number of evaluable cases in each group were 48, 47, and 45, respectively. The non-recurrence rates at 3 years were 54% in group C, 67% in group U, and 85% in group T, and the difference between groups T and C was significant. In terms of the tumor grade and stage, No significant difference was observed among the groups in the category of G1 or Ta tumors, but the non-recurrence rates determined in group T for G2 or T1 tumors were significantly higher than those obtained in group C. Moreover, no significant difference was found among the groups in relation to solitary tumors, but the non-recurrence rate obtained in group T for multiple tumors was significantly higher than that determined in group C. The overall cumulative recurrence rate in each group was 3.07 in group C, 1.95 in group U, and 0.70 in group T, and that determined according to tumor grade, stage, and multiplicity was also highest in group C, followed by group U and group T. The main adverse effects encountered were upper gastrointestinal (GI) symptoms (8.5%) in group U and irritable bladder (11.1%) in group T. Intravesical instillation of thio-TEPA tended to produce greater preventive efficacy than did oral UFT during the early postoperative period, but the prophylactic efficacy of thio-TEPA and UFT should be elucidated over a longer observation period.


Adjuvant chemotherapy with early intravesical instillation of adriamycin and long-term oral administration of 5-fluorouracil in superficial bladder cancer. The Kyushu University Urological Oncology Group.

A randomized controlled trial was performed to study the efficiency of adjuvant chemotherapy with early intravesical instillation of Adriamycin and long-term oral administration of 5-fluorouracil in 275 patients with superficial bladder cancer. All of the patients were randomized into four groups. Group A received early (immediately and 2 days after transurethral resection) instillation of Adriamycin alone; Group B received early instillation of Adriamycin with oral administration of 5-fluorouracil; Group C received delayed (7 days after transurethral resection) instillation of Adriamycin alone; and group D received delayed instillation of Adriamycin with oral administration of 5-fluorouracil. All patients subsequently received instillations weekly for 2 weeks and then every 2 weeks for a further 14 weeks. After 4 months, they received monthly instillations for 8 months. 5-Fluorouracil (groups B and D) was given daily p.o. for 1 year. Evaluation was possible in 187 patients. The postoperative follow-up period for determination of non-recurrence rates was 36 months, during which no significant difference was detected among the four groups. Moreover, no statistically significant difference was found between the early- and delayed-instillation groups. However, the non-recurrence rates obtained in the groups undergoing early instillation were higher than those determined in the delayed-instillation groups during the 36-month follow-up period, and this difference was especially significant at 4 and 5 months. In addition, the early-instillation groups showed significantly higher non-recurrence rates than did the delayed-instillation groups in terms of primary cases (P less than 0.01), tumor size of less than 1 cm (P less than 0.05), multiple tumors (P less than 0.01), pathological stage pTa (P less than 0.01), and histological grades G1 and G2 (P less than 0.05). Groups B and D, which were treated by intravesical instillation of Adriamycin with oral administration of 5-fluorouracil, showed no significant prophylaxis of recurrence during the 36-month follow-up as compared with groups A and C, which received intravesical instillations alone. The main side effect, which required discontinuation of the treatment, was bladder irritation. However, no significant difference in its incidence was found between the early- and delayed-instillation groups. No severe systemic side effect was encountered in this study. These results suggest that early as well as repeated intravesical instillation of Adriamycin is clinically tolerable and may be effective in preventing the recurrence of superficial bladder cancer.


Intravesical combination chemotherapy with mitomycin C and doxorubicin for superficial bladder cancer: a randomized trial of maintenance versus no maintenance following a complete response.

Between November 1986 and April 1989, 101 patients with superficial bladder cancer were treated with intravesical instillations of mitomycin C on day 1 and doxorubicin on day 2 of each week for 5 consecutive weeks. Of 61 complete responders, 23 patients with carcinoma in situ and 28 with papillary cancer were randomly assigned to a non-maintenance group or to a group receiving maintenance therapy consisting of monthly instillations of the same drugs for 12 months. The 2-year non-recurrence rate calculated for patients with carcinoma in situ was significantly better in the maintenance group than in the non-maintenance group. A similar tendency was observed for patients with papillary cancer, although the difference was not significant. Side effects were considerable, with moderate to severe bladder irritation occurring in approximately half of the patients. In addition to our previous findings, the present results indicate that this intravesical combination chemotherapy is effective in eliminating superficial bladder cancers and that since the effect is not durable, even in complete responders, maintenance therapy is necessary to reduce subsequent tumor recurrence.


Sequential instillation therapy with mitomycin C and adriamycin for superficial bladder cancer.

Intravesical chemotherapy involving the sequential instillation of mitomycin C (MMC) and Adriamycin (ADM) was performed in 40 patients with superficial bladder cancer (pathological stages Ta and T1). In all, 20 mg MMC on day 1 and 30 mg ADM on day 2 were instilled into the bladder. This treatment was repeated weekly for 6 consecutive weeks and then monthly for 22 months in cases patients who did not experience serious side effects. A total of 20 patients were treated for multiple recurrences, and the efficacy was evaluated. In all, 9 subjects (45%) achieved a complete response and 6 (30%) showed a partial response, for an overall response rate of 75%. The other 20 patients, including 9 with primary multiple or high-grade tumors and 11 with recurrent tumors, received prophylactic instillation therapy after undergoing transurethral resection (TUR) of their lesions. Of the 9 primary cases, 3 recurred at 19, 8, and 3 months after TUR, respectively, whereas 6 showed no recurrence over a mean follow-up period of 14 months. Of the 11 recurrent cases, the 100-patient-month recurrence rate of 11.9 obtained prior to this treatment fell to 1.4 after the start of therapy. Chemical cystitis was observed in 20 of the 40 patients treated, but the symptoms were transient and tolerable.


Intravesical instillation of adriamycin for bladder tumors.

In the present series of trials, Adriamycin (ADM) intravesical instillation therapy was found to be effective against tumors of papillary morphology measuring less than 10 mm in diameter that were of low pathological stage and low histological grade. The rate of complete disappearance increased in proportion to the concentration of ADM and the duration of retention of the drug, although the efficacy rates were almost the same in each trial. On the other hand, side effects on the bladder were reduced when the instilled solution was lower in concentration and the retention time was short.


Intra-arterial adriamycin chemotherapy in combination with radiotherapy for advanced bladder cancer.

A total of 38 patients with locally advanced bladder cancer (T2, n = 14; T3, n = 14; T4, n = 10) were treated with intra-arterial Adriamycin chemotherapy in combination with radiotherapy. The clinical as well as the pathological efficacy of this treatment was evaluated in all patients. Clinically, 23 (60.5%) of the 38 patients achieved a complete remission (CR), 12 (31.6%) achieved a partial remission (PR), and 3 (7.9%) remained stable (NC). The pathological efficacy was evaluated according to the criteria of Shimosato et al., with 20 (52.6%) of the 38 patients being categorized as grade IV; 2 (5.3%), as grade III; 12 (31.6%) as grade II; and 4 (10.5%), as grade 0. The 5-year actuarial survival as a function of clinical stage amounted to 91.6% for T2, 50.0% for T3, and 37.4% for T4 (T2 vs T3, P less than 0.05; T2 vs T4, P less than 0.01). The 5-year actuarial survival determined according to the clinical and the pathological efficacy of treatment were 74.1% for CRs, 56.2% for PRs, and 0 for NCs (CR vs NC, P less than 0.01; PR vs NC, P less than 0.01). Surgery for preservation of the bladder was performed in 30 of the 33 patients who achieved clinical and pathological CRs or PRs. The 5-year actuarial survival of these 30 patients was 73.2%. These results demonstrate that this therapy is a useful method for the treatment of locally advanced bladder cancer and that preservation of the bladder might be feasible in patients who achieve clinical and pathological CRs or PRs during this treatment.


Intravesical instillation of adriamycin in the presence or absence of verapamil for the treatment of superficial bladder cancer: preliminary report of a collaborative study.

A case-controlled collaborative study on the intravesical administration of Adriamycin in the presence or absence of verapamil, a calcium-channel blocker, as chemotherapy of superficial bladder cancer was carried out at two universities, Okayama and Kagoshima, and their affiliated hospitals. Although little is known about the expression of P-glycoprotein in superficial bladder cancer, it may be a cause of multidrug resistance (MDR). Verapamil was used as an inhibitor of P-glycoprotein. Arm A consisted of Adriamycin given at 50 mg/50 ml saline, and arm B constituted Adriamycin given at 50 mg/40 ml saline plus 5 ampules (10 ml) of injectable verapamil. The drugs were instilled into the bladder for 3 consecutive days in each of 3 consecutive weeks for a total of 9 instillations. No significant difference in antitumor effects was observed between arm A and arm B. Recurrent tumors responded better than did primary tumors to both arm-A and arm-B treatments (P = 0.012). In both treatment arms, significant differences (P = 0.031) in the response rate were found between tumors with diameters of less than 1 cm and those measuring 1-3 cm in diameter. Although the number of evaluable patients was limited, recurrent subjects who had previously received Adriamycin instillations responded in both treatment arms.


Results of adjuvant chemotherapy for invasive uroepithelial cancer.

Between June 1982 and July 1990, 55 patients (41 with bladder cancers and 14 with renal pelvic or ureteral cancers) who had undergone radical extirpative surgery and/or node dissection for pathological stage pT2-4 and/or nodal disease received adjuvant chemotherapy consisting of cisplatin alone or in combination with other agents. In all, 26 of the bladder-cancer patients also received preoperative chemotherapy consisting of arterial infusion of cisplatin, mitomycin C, and Adriamycin. Adjuvant chemotherapy was performed according to the following protocol. Between June 1982 and July 1987, 30-50 mg/m2 cisplatin either alone or in combination with Adriamycin and 5-fluorouracil (CAF) was given to 35 patients in an induction and maintenance setting for 1 year. After July 1987, short-course cisplatin (70 mg/m2) or cisplatin, etoposide, and Adriamycin combination chemotherapy (CVA) was given to 20 patients. Of the 55 patients, 38 are alive and show no evidence of disease, three are alive with disease, 13 have died of their disease, and 1 has died of an unrelated cause. The 5-year survival of all patients was 65.1%. The survival of the 20 patients who were treated after July 1987 was better than that of the 35 patients who were treated before June 1987. Local recurrence and/or distant dissemination occurred in 16 patients, 13 of whom died of cancer progression. Nausea and vomiting and anorexia occurred in most patients during the administration of cisplatin. Mild to moderate myelosuppression developed in patients who received CAF or CVA combination chemotherapy. Although adjuvant chemotherapy combined with radical surgery seemed to be effective in cases with a pathological stage of pT3a or less, more intensive pre- or postoperative chemotherapy is needed to improve the poor prognosis of patients with deeply invasive uroepithelial cancer.


Combination chemotherapy for advanced urothelial-tract carcinoma.

Between December 1982 and November 1990, 31 patients with advanced urothelial carcinoma were treated with one of two combination chemotherapy regimens. A total of 20 patients were treated with 3 mg/m2 mitomycin C and 300 mg/m2 cyclophosphamide given intravenously every 10-14 days and with 180 mg/m2 5-fluorouracil (5-FU) given intravenously every day for as long as possible (CF-Mito regimen). After the patient had been discharged from the hospital, the same treatment with CF-Mito was performed except that 180 mg/m2 5-FU was replaced by 400 mg/m2 UFT (a mixture of tegafur and uracil) given orally. A total of 11 patients whose tumor had relapsed during the first-line treatment were given 60 mg/m2 cisplatin, 40 mg/m2 Adriamycin, and 40 mg/m2 methotrexate intravenously every 28 days (PAM regimen). In all, 20 patients received 4-44 (mean, 9.7) courses of CF-Mito over a period of 1.5-24 (mean, 5.3) months. The results obtained in these 20 patients with evaluable lesions included no complete remission (CR), 4 partial remissions (PRs), 9 cases of stable disease (SD), and 7 cases of progressive disease (PD). The PR duration was 1.5-22 (mean, 7.5) months. The side effects encountered in this group included anorexia, nausea, vomiting, myelosuppression, diarrhea, stomatitis, liver damage, and heart failure. In all, 11 patients received 3-7 (mean, 4.1) courses of PAM over a period of 3-14.5 (mean, 5.2) months. All 11 patients had evaluable lesions, and their responses included no CR, 5 PRs, 3 cases of SD, and 3 cases of PD. The PR duration was 1-3 (mean, 1.6) months. The side effects encountered in this group included anorexia, nausea, vomiting, myelosuppression, heart failure, and hair loss.


Use of methotrexate, vinblastine, adriamycin, and cisplatin in combination with radiation and hyperthermia as neo-adjuvant therapy for bladder cancer.

In an attempt to improve the poor prognosis of invasive and/or high-grade bladder cancer after total cystectomy, we tried a combination of regional irradiation with hyperthermia (RH) therapy and systemic M-VAC (methotrexate, vinblastine, Adriamycin, and cisplatin) chemotherapy followed by surgery. The short-term results of these treatments were evaluated. A total of 17 patients received the combination of RH and M-VAC therapy between January 1989 and July 1990, and 12 then underwent total cystectomy. Of the 17 patients, 14 were evaluable for tumor response. The objective response rate was 64% (9/14), with 4 patients achieving a complete remission that was confirmed by histological examination. Nausea and vomiting were inevitable, and 71% (12/17) of the patients developed leukopenia. However, these side effects were not serious. Considering the previous results obtained using RH therapy in the absence of chemotherapy for this disease, no significant difference in the tumor response was detected between the RH only group and the RH plus M-VAC group. The long-term results cannot yet be evaluated, but we will continue to follow these patients in the future so as to clarify the usefulness of M-VAC therapy as preoperative therapy.


Clinical and pathological evaluations of methotrexate, vinblastine, adriamycin and cisplatin chemotherapy for advanced urothelial cancers.

We have treated advanced transitional-cell carcinoma of the urothelial tract with methotrexate, vinblastine, Adriamycin, and cisplatin (M-VAC) chemotherapy since July of 1985. We analyzed the effect of that chemotherapy in 26 patients with advanced urothelial cancer who were treated in our hospital and followed up. They were divided into two groups. Group 1 consisted of 15 patients with distant metastases. In all, 11 of them received M-VAC as adjuvant chemotherapy for metastatic lesions after surgical removal of the primary lesion, and the remaining 4 patients were not operable since they had very advanced-stage tumors; they received only M-VAC chemotherapy. Group 2 contained 11 patients who received M-VAC neo-adjuvant chemotherapy. In group 1, the overall response rate was 57.1% and the mean duration of response was 12.6 months. In the 11 patients who had received M-VAC as adjuvant therapy after surgical removal of the primary tumor, the mean duration of response was 14.1 months. After M-VAC chemotherapy, six patients underwent surgical resection of metastatic lesions and restaging was done pathologically in these cases. The clinical response coincided with the pathological response in all six cases. In group 2, 5 of 11 patients experienced histological downstaging of the resected bladder. M-VAC chemotherapy combined with surgical resection of residual tumors has proved to be an effective option against advanced urothelial cancer.


Methotrexate, vinblastine, doxorubicin, and cisplatin for advanced urothelial cancer.

A series of 31 patients with advanced urothelial cancer were treated with combination chemotherapy consisting of 1-4 cycles of methotrexate, vinblastine, doxorubicin, and cisplatin (M-VAC). Of the 31 patients, 29 had measurable and evaluable lesions. A complete remission was achieved by 4 of these 29 patients (14%) for 1-46 months. A partial remission was observed in 14 of the 29 patients (48%) for 1-9 months. Whereas bony and hepatic metastatic lesions did not respond, some nodal (7/12), pulmonary (4/8), and pelvic lesions (2/3) as well as primary bladder tumors (4/6) and a tumor marker (1/2) responded. Complete tumor remission was observed in nodal (2/12) and pulmonary (1/8) metastatic lesions, in invasive lesions to the prostate and seminal vesicle (1/1), and in primary lesions in the bladder (2/6), ureter (1/1), and urethra (1/1). Two of three patients with non-transitional cell tumors attained a partial remission for 1-7 months. Complete remission of the pulmonary lesions was obtained in a case of squamous cell cancer of the bladder with pulmonary metastases. The toxicity of this regimen was generally tolerable and included moderate to severe myelosuppression, mild to moderate nausea and vomiting, renal toxicity, and mucositis. These results suggest that the M-VAC regimen holds promise for the treatment of advanced metastatic transitional cell cancer as well as non-transitional cell cancer of the urothelium.


Factors affecting the outcome of patients with advanced urothelial cancer following chemotherapy with methotrexate, vinblastine, adriamycin, and cisplatin.

Attempts were made to identify factors related to the response of patients treated with intravenous methotrexate, vinblastine, Adriamycin, and cisplatin (M-VAC). The subjects consisted of 54 patients with advanced urothelial cancer whose histological type was transitional-cell carcinoma. The effects of various factors on the response were studied using univariate analysis and a multiple logistic regression model. The following factors were included in the analyses: (1) age, (2) sex, (3) performance status (PS), (4) primary site, (5) histological grade, (6) T category, (7) N category, (8) M category, (9) tumor status, and (10) dose of drugs. In all, 9 patients achieved a complete response and 23 showed a partial response, for an overall response rate of 59% (95% confidence limits, 46%-72%). Univariate analysis revealed that the PS, M category, and dose of drugs were related to the response, and there was a significant correlation among these three factors. In the multiple logistic regression model, the absolute value of t was high for the M category. The presence of distant metastases is an important factor in predicting poor efficacy for the present regimen. The management of metastatic disease will be the subject of further study in the treatment of advanced urothelial cancer.


Cisplatin-based chemotherapy for the treatment of advanced transitional-cell carcinoma of the urinary tract--a preliminary report.

The CMV (cisplatin, methotrexate, and vinblastine) and M-VAC (methotrexate, vinblastine, doxorubicin, and cisplatin) regimens were used to treat 19 patients with advanced transitional-cell carcinoma (TCC) of the urothelial tract. In the CMV group, the partial response rate was 45.5% and the mean response duration was 6.3 months. No complete response was obtained in our series. The median duration of survival was 15.8 and 8.3 months in responders and nonresponders, respectively. The toxic symptoms included one case of sepsis and three cases of renal toxicity. However, nausea and vomiting were experienced by most patients and required the administration of antiemetics. In the M-VAC group, the median duration of survival for responders was longer than that of nonresponders (greater than 10.2 vs 7.2 months), although the number of patients was too small for this difference to reach statistical significance. The toxic symptoms included one case of sepsis, two cases of renal toxicity, and nausea and vomiting in most patients. Bone metastasis in three patients did not respond to chemotherapy (CMV), a finding that is compatible with the results reported by other investigators. In summary, chemotherapy with the CMV or M-VAC regimen was effective in improving the response rate of patients. However, the duration of response was short, toxicity was severe in some cases, and the efficacy against bone lesions was poor. These problems must be solved to improve the outcome of patients with TCC following chemotherapy with the CMV or M-VAC regimens.


Evaluation of systemic chemotherapy with methotrexate, vinblastine, adriamycin, and cisplatin for advanced bladder cancer. The Japanese Urological Cancer Research Group for Adriamycin.

In a cooperative study of the Japanese Urological Cancer Research Group for Adriamycin, the usefulness of chemotherapy with methotrexate, vinblastine, Adriamycin, and cisplatin (M-VAC therapy) in treating advanced or recurrent bladder cancer was examined. Evaluation of the clinical responses obtained in 86 evaluable patients revealed 13 complete responses, 29 partial responses, 4 minor responses, 19 cases of no change, and 21 cases of progressive disease. The overall response rate was 48.8% (42/86). The rate of response to M-VAC therapy at each disease site was as low as 21.4% (3/14) in bone lesions but exceeded 40% in the primary lesion, the lymph nodes, the lung, the liver, and other lesions. The clinical response to M-VAC therapy was not significantly influenced by the performance status of the patients, the dose intensity, or previous therapy. The median duration of response for the 42 responders was 22.7 weeks (range, 8.1-134.1 weeks), and the median duration of survival for the 86 evaluable patients was 9.8 months. Side effects were frequently encountered; the patients experienced anorexia, nausea, vomiting, malaise, alopecia, and leukopenia, but all of these symptoms were tolerable.


The use of adriamycin and its derivatives in the treatment of prostatic cancer.

Adriamycin and the other anthracyclines are amongst the most effective cytotoxic agents at the clinician's disposal. At least one of the reasons for their efficacy is the large number of mechanisms by which they can induce potentially lethal damage in a dividing malignant cell. Adriamycin and epirubicin are amongst the more effective agents in advanced hormone-independent prostate cancer. When given in full doses, they produce a reasonable number of objective as well as subjective responses, but the resultant toxicity precludes their use at high dose in many patients. In a number of well-documented studies, lower doses of these agents have yielded useful subjective responses with minimal toxicity.


Intravesical therapy with adriamycin and 4-epirubicin for superficial bladder cancer: the experience of the EORTC GU Group.

The anthracycline derivatives Adriamycin and 4-epirubicin are used to prevent recurrent tumors after transurethral resection of superficial bladder tumors. Both drugs are instilled intravesically. The present report describes the results of two multicenter, prospective, randomized phase III studies carried out by the EORTC GU Group. In protocol 30,790, after a mean follow-up period of 26.6 months, the recurrence rate for 165 patients treated with Adriamycin was 0.29 and the tumor rate was 0.74. For 156 patients treated with Epodyl, the recurrence rate was 0.29 and the tumor rate was 0.57. This difference was not statistically significant. For 70 patients who received transurethral resection alone, the recurrence rate was 0.65 and the tumor rate, 2.04. In protocol 30,763, patients with good prognostic factors were treated with one single instillation of 4-epirubicin versus sterile water. After a mean follow-up period of 16 months, in 190 patients treated with 4-epirubicin the recurrence rate was 0.20 and the tumor rate was 0.35; in 196 patients treated with sterile water, the recurrence rate was 0.37 and the tumor rate was 0.65 (P = 0.0001). Adriamycin and 4-epirubicin were efficacious, and severe side effects were not encountered. The superiority of Adriamycin over Epodyl could not be proven.


Caffeic acid causes metal-dependent damage to cellular and isolated DNA through H2O2 formation.

Pulsed field gel electrophoresis showed that caffeic acid induced DNA strand breaks in cultured human cells in the presence of Mn(II). With alkali treatment, DNA single-strand breaks were observed. The strand breakage was increased by the treatment of buthionine sulphoximine (a GSH synthesis inhibitor) and 3-aminotriazol (a catalase inhibitor) and decreased by catalase, indicating the involvement of H2O2. The DNA damage was decreased by o-phenanthroline, indicating the involvement of transition metal ion. Damage to isolated DNA from c-Ha-ras-1 protooncogene was investigated by a DNA sequencing technique. Caffeic acid caused DNA damage in the presence of Cu(II) but not in the presence of either Mn(II) or Fe(III). Caffeic acid plus Cu(II) induced piperidine-labile sites frequently at thymine residues, especially of the 5'-GTC-3' and 5'-CTG-3' sequences. Typical OH scavengers showed no inhibitory effects. The inhibitory effects of bathocuproine and catalase on Cu(II)-mediated DNA damage suggest that Cu(I) and H2O2 have important roles in the production of active species causing DNA damage. The Cu(II)-mediated DNA damage was enhanced by pre-incubation of caffeic acid with Mn(II). Mn(II)- or Cu(II)-catalyzed autoxidation of caffeic acid produced H2O2 with efficiency of Mn(II) greater than Cu(II). These results suggest that in the presence of Mn(II) or Cu(II), caffeic acid produces H2O2, which is activated by transition metals to cause damage to DNA in vitro and probably in cultured cells.


Chemotherapy of advanced transitional-cell carcinoma of the bladder.

A number of single agents and multidrug combinations are useful in the therapy of advanced transitional-cell carcinoma of the bladder. Phase II studies have identified cisplatin, Adriamycin (doxorubicin), methotrexate, and vinblastine as the most active cytotoxic agents. Combination chemotherapy based on cisplatin has shown greater efficacy than older regimens based on Adriamycin or methotrexate. Trials of regimens containing both cisplatin and methotrexate, such as those conducted by the Northern California Oncology Group using CMV (cisplatin, methotrexate, and vinblastine), have reported that a significant number of patients respond to treatment, with frequent complete responses being noted. Anthracycline-containing regimens such as M-VAC (methotrexate, vinblastine, Adriamycin, and cisplatin) have also played an important role in the therapy of advanced bladder cancer. Trials comparing cisplatin- and methotrexate-containing regimens with single-agent cisplatin or other cisplatin combinations have shown the apparent superiority of the former in terms of greater overall response rates and improved survival. However, the toxicity of such regimens can be significant, and phase III studies are under way to validate their use in the neoadjuvant setting.


O6-methylguanine-DNA methyltransferase activity in human tumors.

The distribution of O6-methylguanine-DNA methyltransferase (MGMT) activity in extracts of tumors from 74 patients was measured. The results demonstrated that there was considerable variation of MGMT activity in different human tumor tissues as well as in different individuals. The mean values (X +/- SD, pmol/mg of protein) in breast cancer, stomach cancer, small cell lung cancer, non-small cell lung cancer, renal cell carcinoma, esophageal carcinoma, brain tumors, colon carcinoma and malignant melanoma were 1.071 +/- 0.374 (9), 0.515 +/- 0.107 (5), 0.509 +/- 0.251 (5), 0.461 +/- 0.227 (24), 0.329 +/- 0.246 (5), 0.273 +/- 0.376 (5), 0.244 +/- 0.175 (14), 0.242 +/- 0.308 (5) and 0.201 +/- 0.161 (2) respectively. It was notable that six samples (1/24 non-small cell lung cancer, 3/5 esophageal carcinoma, 1/14 brain tumors and 1/5 colon carcinoma) did not have any detectable level of MGMT activity. Activity of glutamine pyruvic transaminase (GPT) was also measured in the same extracts used for the assay of MGMT activity. The activity of GPT in these samples with undetectable level of MGMT activity was similar to those with significant MGMT activity. These results further strengthen the assumption that a certain fraction of human tumors are Mer-.


Aberrant crypts correlate with tumor incidence in F344 rats treated with azoxymethane and phytate.

Aberrant crypts are putative preneoplastic lesions that have been proposed as intermediate biomarkers for colon cancer. The goals of these studies were to determine (i) if the colon cancer chemopreventive agent, sodium phytate, when started 1 week after a single dose of carcinogen, has any effect on the development of aberrant crypt foci (ACF) in treated rats; and (ii) if ACF at an early time period under these conditions correlate with the later formation of tumors in similarly treated animals. The number of ACF with four or more crypts was greater (P = 0.02, Mann-Whitney test) in rats with tumors compared with rats without tumors killed at 36 weeks after the injection of azoxymethane (AOM); the total number of ACF was not significantly different in these two groups. The incidence of tumors in F344 rats treated with AOM without phytate was 83% (10/12) compared to 25% (3/12) in rats treated with AOM plus phytate (P = 0.0045, two-tail Fisher's exact test). The finding of more (P = 0.005, Mann-Whitney test) ACF with four or more crypts in rats without phytate than in rats with phytate at 12 weeks after the injection of AOM is consistent with the hypothesis that the development of larger ACF (with four or more crypts) is predictive of the tumor incidence. These results validate the use of this parameter, i.e. ACF with four or more crypts, as an intermediate biomarker for tumor incidence in this system.


Enhancing effects of diallyl sulfide on hepatocarcinogenesis and inhibitory actions of the related diallyl disulfide on colon and renal carcinogenesis in rats.

It has been reported that diallyl sulfide (DS) and diallyl disulfide (DDS), major volatile compounds in garlic (Allium sativum), exert anticarcinogenic activity in several organs in rodents. The modifying effects of these two chemicals were therefore assessed using two-step liver and multi-organ carcinogenesis models. In experiment 1, male F344 rats were given a single i.p. injection of N-diethylnitrosamine (200 mg/kg body wt) and then received DS or DDS by intragastric intubation at doses of 200 and 50 mg/kg body wt, respectively, three times a week for 6 weeks. All rats were subjected to two-thirds partial hepatectomy at experimental week 3. In experiment 2, male F344 rats were sequentially treated with five carcinogens with different organ target sites for 4 weeks, and then administered DS or DDS as in experiment 1 for 24 weeks. DS demonstrated clear enhancing effects on the development of glutathione S-transferase placental form positive foci in both experiments. On the other hand, an inhibitory potential in colon and renal carcinogenesis was observed in rats treated with DDS. Therefore, while DDS may act as a chemopreventive agent, DS may promote hepatocarcinogenesis.


Azaserine-induced pancreatic foci: detection, growth, labelling index and response to raw soya flour.

Atypical acinar cell foci were induced in the pancreases of rats by injection of azaserine. An incubation period of 6 weeks was sufficient for the detection of all glutathione S-transferase mu positive foci. In chow-fed rats, the labelling index of foci was 12-fold higher than normal pancreatic tissue. Feeding rats raw soya flour (RSF) for up to 20 weeks did not increase the number of foci per pancreas but did produce significant increases in labelling index and growth rate. In normal pancreatic tissue, the trophic response was complete after 4 weeks of RSF feeding. In foci, however, the trophic response to RSF was prolonged. Involution of normal pancreatic tissue was seen in rats fed RSF for 19 weeks and then switched to chow 1 week prior to death. No evidence for involution was seen in the foci of these animals, although a 40-fold reduction was seen in labelling index. The labelling index of these foci was reduced to the level seen in normal tissue of chow-fed rats. These results are consistent with increased cholecystokinin (CCK) responsiveness and CCK dependence in azaserine-induced pancreatic foci.


Effects of cholecystokinin and bombesin on development of azaserine-induced pancreatic tumours in rats: modulation by the cholecystokinin receptor antagonist lorglumide.

Cholecystokinin and bombesin have been shown to promote pancreatic growth and development of azaserine-induced acidophilic atypical acinar cell nodules in rat pancreas after treatment for 16 weeks. Lorglumide, a specific cholecystokinin receptor antagonist, inhibited the stimulating effect of cholecystokinin, but not of bombesin. The present study was carried out to determine effects of cholecystokinin and bombesin, alone and in combination with lorglumide, on pancreatic growth and carcinogenesis after chronic treatment. The animals were killed 8 months after the start of treatment. Growth of the pancreas and the development of acidophilic atypical acinar cell nodules in exocrine pancreas was enhanced significantly by both cholecystokinin and bombesin, but the number of carcinomas was increased only by bombesin. Lorglumide inhibited the effects of cholecystokinin on both pancreatic growth and on the development of acidophilic nodules. The effects of bombesin on pancreatic growth and development of pancreatic lesions, except for adenomas, were not inhibited by lorglumide.


Effect of carcinogen dose and age at administration on induction of mammary carcinogenesis by 1-methyl-1-nitrosourea.

The objective of the work reported in this paper was to determine if the tumorigenic response to 1-methyl-1-nitrosourea (MNU) in the mammary gland varied with age of administration and was dose dependent when the carcinogen was injected prior to 50 days of age. Using a recently developed method for mammary tumor induction, MNU was injected i.p. at doses ranging from 25 to 75 mg/kg at 35 days of age or 50 mg/kg at 28, 35 or 42 days of age. Treatment with MNU resulted in induction of both benign and malignant mammary tumors. The incidence of mammary gland adenocarcinomas was 100% at and above the 50 mg/kg dose of MNU, irrespective of the age at which carcinogen was administered. The number of cancers increased in proportion to carcinogen dose, whereas cancer latency decreased as the MNU dose increased. In rats injected with 50 mg MNU/kg body weight at 28, 35 or 42 days of age, differences among groups in cancer incidence, number or latency were not statistically significant. Metastases of mammary neoplasms to lung, liver and spleen were observed in rats injected with MNU at 35 or 42 days of age. These data indicate (i) the dose responsiveness of MNU-induced mammary carcinogenesis in rats initiated prior to 50 days of age; (ii) the lack of effect of age at initiation if prior to 50 days on final tumor outcome; and (iii) that the age at which MNU is injected may affect the metastatic potential of the mammary carcinomas that are induced.


Progressive atypia in spontaneous and N-nitrosodiethylamine-induced hepatocellular adenomas of C3H/HeNCr mice.

The progression of hepatocellular adenomas to carcinomas has been less well documented in mice than in rats. We studied progression of spontaneous and chemically induced hepatocellular adenomas in male C3H/HeNCr mice by image analysis. Spontaneous lesions in 15, 18 and 21 month old untreated male C3H/HeNCr mice and experimentally induced lesions were examined. Experimental group 1 received a single i.p. injection of N-nitrosodiethylamine (DEN) (5 mg/kg body wt) at 15 days of age. Groups 2 and 3 were injected a second time with DEN at 15 or 20 weeks of age (75 mg/kg body wt), with interim sacrifices at 11, 16 and 34 weeks after the second DEN injection. Atypia in adenomas were classified into four grades according to cell size, tinctorial changes, cellular pleomorphism and trabecular pattern. At earlier stages of the neoplastic process (11 or 16 weeks after the second DEN dose), most adenomas were well-differentiated lesions with no atypia or focal grade 1 or 2 atypia. At later stages (34 weeks after the second DEN dose), a large proportion of hepatocellular tumors were classified as adenoma with grade 3 atypia or carcinoma. The proportion of carcinomas in mice treated with a second dose of DEN at 20 weeks of age was significantly higher than in mice treated with a single dose of DEN or in mice given a second dose of DEN at 15 weeks. A positive correlation was found between increase in the size of lesions and increased atypia in both spontaneous and DEN-induced lesions and with age for spontaneous tumors. These results support the hypothesis that mouse hepatocellular adenomas are truly neoplastic lesions in different stages of progression toward malignancy.


Glutathione conjugation of trans-3,4-dihydroxy 1,2-epoxy 1,2,3,4-tetrahydrobenzo[c]phenanthrene isomers by human glutathione transferases.

Each of the four stereoisomers of trans-3,4-dihydroxy 1,2-epoxy 1,2,3,4-tetrahydrobenzo[c]phenanthrene [(+)- and (-)-anti-BPhDE and (+)- and (-)-syn-BPhDE] has been incubated with the human glutathione transferase (GST) isoenzymes GST A1-1, GST M1-1 and GST P1-1, representing class alpha, mu and pi respectively, and glutathione (GSH). The conjugates formed were analyzed by HPLC and the results demonstrate that all GST isoenzymes catalyze the formation of GSH conjugates of all BPhDE isomers. However, a marked variation in catalytic efficiencies was observed (0.122-1.28/mM/s). These values are considerably lower than those previously estimated for the bay-region diol epoxides of benzo[a]pyrene (B[a]P) and human GSTs. The (+)-syn and (-)-anti-BPhDE (1R,2S-epoxide absolute configuration) were in general better substrates than the corresponding 1S,2R-epoxides. In accordance with previous observations with the diolepoxides of B[a]P, GST P1-1 was highly selective towards the BPhDE isomer with 4R,3S-diol 2S,1R-epoxide absolute configuration, i.e. (-)-anti-BPhDE, whereas GST A1-1 and M1-1 preferentially catalyzed the conjugation of (+)-syn-BPhDE (4R,3S-diol 2R,1S-epoxide absolute configuration). Overall, the most active isoenzyme was GST A1-1. Analysis by NMR spectroscopy of the GSH conjugates of BPhDE demonstrate that the reaction with GSH generally takes place by trans-addition of the thiol group at the benzylic C-1 carbon. The low catalytic efficiencies of human GSTs with BPhDE as compared to diolepoxides of B[a]P may be explained in part by the more crowded bay-region and substantially lower chemical reactivity (e.g. delta Edeloc/beta) of the former compounds.


Endogenous glutathione levels modulate the frequency of both spontaneous and long wavelength ultraviolet induced mutations in human cells.

Spontaneous and induced mutations at the hypoxanthine guanine phosphoribosyl transferase locus have been measured in cultured human lymphoblastoid (TK6) cell populations under conditions in which cellular glutathione has been severely depleted by overnight treatment with buthionine-S,R-sulfoximine. At maximum levels of glutathione depletion, the increase in spontaneous frequency is at least 5-fold, a finding consistent with the possibility that cellular redox state can modulate the levels of pre-mutagenic damage arising as a result of normal metabolism in cultured human cells. Glutathione depletion does not lead to a significant enhancement in the frequency of mutants that arise as a result of irradiation at 313 nm but does lead to a 3-fold increase in mutations resulting from irradiation at 365 nm. These results indicate that glutathione may quench reactive intermediates that would otherwise lead to spontaneous mutations as well as a fraction of UVA radiation-induced premutagenic damage.


Foreign compound metabolism capacity in man measured from metabolites of dietary caffeine.

Caffeine is sequentially metabolized by cytochrome P4501A2 (CYP1A2), N-acetyltransferase (NAT) and/or xanthine oxidase (XO). In the present study the activity of these three enzymes was estimated from ratios of the metabolites formed from dietary caffeine and excreted into the urine collected as spot samples. In the urine samples from 10 out of 377 subjects concentrations of caffeine metabolites were too low to allow reliable measurements of the ratios. In 335 healthy subjects the NAT activity showed a typically bimodal distribution with 47% fast acetylators and 53% slow acetylators, consistent with a Danish population. The ratios reflecting CYP1A2 and XO activities were log normal and normal distributed, respectively. In 103 non-smoking men and 90 non-smoking women the ratio of caffeine metabolites expressing CYP1A2 activity was 4.7 +/- 1.6 and 4.3 +/- 1.9 as compared to 7.8 +/- 2.5 and 7.3 +/- 3.0 in 31 male and 25 female subjects smoking 10 cigarettes/day or more respectively, verifying induction of CYP1A2 by tobacco (P less than 0.05), but minimal sex-related differences. In 12 non-smoking pregnant women and in 28 women using oral contraceptives the CYP1A2 ratio was 29 and 20% reduced respectively (P less than 0.05). In a multivariate analysis the only significant predictor of the XO ratio was the consumption of caffeine with an increase of 2% per cup of coffee or equivalent (P less than 0.05). In 23 healthy male subjects 30 days of vigorous exercise increased the CYP1A2 ratio by 70% and the XO ratio by 42% (P less than 0.05), but left the NAT ratio unchanged. In nine healthy volunteers daily ingestion of 500 g of broccoli for 10 days increased the CYP1A2 ratio by an average of 12% (P less than 0.05), compared to a control period with ingestion of an equivalent weight of non-cruciferous green vegetables. The ratios of metabolites from dietary caffeine in spot urine samples offer ethical, non-invasive and reliable estimates of CYP1A2, NAT and XO. These enzymes are highly relevant for the bioactivation of potentially toxic compounds and the formation of oxygen radicals. The method is applicable in large-scale epidemiological studies, allowing, for example, prospective testing of the relationship between these enzyme activities and the development of disease. Exercise may increase CYP1A2 activity to a magnitude corresponding to heavy smoking, as well as XO by mechanisms that remain to be clarified.


Coal tar therapy does not influence in vitro benzo[a]pyrene metabolism and DNA adduct formation in peripheral blood lymphocytes of psoriatic patients.

Human lymphocytes (HL) from healthy subjects and psoriatic patients were treated in vitro for 24 h with 4 microM [3H]benzo[a]pyrene (B[a]P) and 2 microM (-)-B[a]P-7,8-dihydrodiol in order to verify if the coal tar (CT) used in the therapy of psoriasis, which is characterized by a high content of polycyclic aromatic hydrocarbons (PAH), influences the formation of B[a]P-DNA adducts and the metabolism of B[a]P. Significant amounts of syn-BPDE-dGuo adducts were detected in all the examined HL samples, but no significant difference in the amounts of total BPDE-DNA adducts or of specific anti- and syn-BPDE-dGuo adducts was observed between healthy subjects and psoriatic patients, or between psoriatic patients before and after CT treatment. Moreover, the CT treatment of psoriatic patients did not influence the enantiomeric composition of B[a]P-7,8-dihydrodiols present in the HL culture medium, among which the (-)-7R,8R form was found to predominate (greater than 98%) in all the HL samples. The existence in HL of a specific metabolic pathway of B[a]P leading to the formation of (-)-syn-BPDE and the corresponding tetrols, through the epoxidation of the (-)-7R,8R enantiomer of B[a]P-7,8-dihydrodiol, was confirmed by determining the tetrols derived from the syn- and anti-stereoisomers of BPDE, released in HL culture medium after treatment for 24 h with 2 microM (-)-B[a]P-7,8-dihydrodiol. Although B[a]P-7,10/8,9 and B[a]P-7/8,9,10 tetrols, derived from (+)-anti-BPDE, were the predominant isomers, significant amounts of B[a]P-7,9/8,10 and B[a]P-7,9,10/8 tetrols, derived from the hydrolysis of (-)-syn-BPDE, were also detected. The mean ratio of anti/syn tetrols in healthy subjects was significantly lower than in psoriatic patients, but no difference in that ratio was found in psoriatic patients before and after CT treatment.


Contribution of dihydrodiol dehydrogenase to the metabolism of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene in fortified rat liver subcellular fractions.

Dihydrodiol dehydrogenase (DD; EC 1.3.1.20) purified to homogeneity from rat liver cytosol will catalyze the NAD(P)(+)-dependent oxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-diol) to yield benzo[a]pyrene-7,8-dione (BPQ). To verify that BPQ is a metabolite of B[a]P-diol in rat liver, an S100 fraction was supplemented with NAD+ and NADP+, and the formation of BPQ was followed by reverse-phase HPLC. The identity of BPQ was established by co-chromatography with an authentic standard (under different solvent conditions) and by RP-HPLC using a diode-array detector which established that the metabolite shared spectral identity with BPQ. The formation of BPQ in the S100 fraction was blocked by either a competitive inhibitor (indomethacin) or a suicide substrate [1-(4-nitrophenyl)-propen-1-ol] for DD, indicating that BPQ was being formed by this enzyme. To assess the contribution of DD to the metabolism of [3H]B[a]P-diol, subcellular fractions obtained from uninduced rat liver were fortified with co-factors to optimize the activity of enzymes that would compete for this proximate carcinogen. Under these conditions, S100 fractions fortified with NAD+ and NADP+ metabolized 25% of the B[a]P-diol, producing 731 +/- 154 pmol of BPQ. In contrast, rat liver microsomes fortified with an NADPH generating system metabolize 75% of the B[a]P-diol producing 2614 +/- 379 pmoles of benzo[a]pyrene-tetrahydrotetrols. Rat liver homogenates (S10) fortified with either uridine diphosphoglucuronic acid or phosphoadenosine phosphosulfate produced 180 +/- 56 and 95 +/- 31 pmoles of conjugates respectively, which were recovered as B[a]P-diol after treatment of the aqueous phase with either beta-glucuronidase or aryl sulfatase. Of the metabolites analyzed BPQ was formed in the second largest amount. These studies show that in uninduced rat liver DD may play a significant role in the metabolism of B[a]P-diol. The metabolic fate of BPQ remains to be determined.


Mutagenesis of the K-ras protooncogene in mouse lung tumors induced by N-ethyl-N-nitrosourea or N-nitrosodiethylamine.

The role of ras gene activation in the development of lung tumors induced by N-ethyl-N-nitrosourea (ENU) and N-nitrosodiethylamine (DEN) was evaluated in the A/J mouse, a strain susceptible to chemically induced lung tumors. DNAs isolated from both ENU- and DEN-induced lung tumors were screened for activating mutations in the K-ras gene by utilizing the polymerase chain reaction (PCR) and direct sequence analysis. Mutations in the K-ras gene were detected in 11 of 11 ENU-induced tumors and 23 of 28 DEN-induced tumors. In ENU-induced tumors, there were three GC----AT transitions in the second base of codon 12, and seven AT----GC transitions and one AT----TA transversion in the second base of codon 61. A similar spectrum of K-ras mutations was observed in DEN-induced lung tumors: five GC----AT transitions and two GC----TA transversions in the second base of codon 12, and sixteen AT----GC transitions at the second base of codon 61. Ninety-one percent (31/34) of the observed mutations are consistent with the formation of the promutagenic O4-ethylthymine and O6-ethylguanine adducts in DNA. Therefore, lung tumors from the A/J mouse induced by DEN and ENU could be initiated by the interaction of reactive metabolites with specific sites in the K-ras gene. This is the first clear example of activation of the K-ras gene by ethylating agents in a rodent lung tumor system.


Characterization of 4,4'-methylenebis(2-chloroaniline)--DNA adducts formed in vivo and in vitro.

4,4'-Methylenebis(2-chloroaniline) (MOCA) is a genotoxic and carcinogenic industrial chemical to which there is considerable potential human exposure. Since metabolic activation and formation of DNA adducts are believed to be important for the induction of these effects, DNA was treated in vitro with radiolabeled N-hydroxy-MOCA, the presumed proximate carcinogenic metabolite formed in vivo. Two major radioactive peaks were observed after HPLC separation of enzymatic hydrolysates. The two products were analyzed by MS and characterized as N-(deoxyadenosine-8-yl)-4-amino-3-chlorobenzyl alcohol and N-(deoxyadenosin-8-yl)-4-amino-3-chlorotoluene. The same adducts were also the major adducts formed in DNA of tissues from rats treated with radiolabeled MOCA. They were eliminated from rat liver with non-linear kinetics, in agreement with observations made for other carcinogens. The selective reaction of N-hydroxy-MOCA with DNA-adenine and the formation of single arylamine ring adducts suggest a substitution mechanism involving an intermediate with strong SN1 character, aided by the negative inductive effect of the ortho-chlorine. Due to tautomer formation, the initial adduct may be inherently unstable and undergo cleavage at the 1'-carbon-methylene bond to yield the observed adducts.


Examination of microsomal cytochrome P450-catalyzed in vitro activation of o-phenylphenol to DNA binding metabolite(s) by 32P-postlabeling technique.

It has been previously reported that the reactive metabolites phenylsemiquinone and phenylbenzoquinone are generated during microsomal cytochrome P450-catalyzed redox cycling of o-phenylphenol (OPP). However, covalent modification of DNA by OPP-reactive metabolites has yet not been demonstrated. In the present study we have investigated the covalent binding in DNA by OPP-reactive metabolites using 32P-postlabeling. Analysis of adducts by 32P-postlabeling in products of chemical reaction of DNA with phenylbenzoquinone revealed four major and several minor adducts. The chemical reaction of deoxyguanosine 3'-phosphate with phenylbenzoquinone also showed four major adducts. The chromatographic mobility of major adducts of deoxyguanosine 3'-phosphate-phenylbenzoquinone was identical to that of major adducts of DNA-phenylbenzoquinone. The major adducts are demonstrated to be stable. The total covalent binding in deoxyguanosine 3'-phosphate by phenylbenzoquinone (686,000-687,000 amol/nmol nucleotide) was higher than that observed in DNA (26,500-28,000 amol/nmol nucleotides). Reaction of DNA with OPP or a hydroxylated metabolite of OPP, phenylhydroquinone, in the presence of microsomes and NADPH or cumene hydroperoxide showed four major adducts. Adduct formation in DNA by OPP or phenylhydroquinone in the presence of the microsomal activation system was drastically decreased by known inhibitors of cytochrome P450. The chromatographic mobility of major adducts in DNA by OPP or phenylhydroquinone in the presence of microsomal activation system matched with those major adducts observed in deoxyguanosine 3'-phosphate or DNA reacted with pure phenylbenzoquinone. These data demonstrate that OPP or phenylhydroquinone, a hydroxylated metabolite of OPP, is able to bind covalently to DNA in the presence of a microsomal cytochrome P450 activation system. Phenylbenzoquinone is one of the DNA-binding metabolite(s) of OPP. It is concluded that OPP is genotoxic in an in vitro system and genotoxicity produced by OPP-reactive metabolites may play a role in OPP-induced cellular toxicity or cancer.


Spectrum of mutations in single-stranded DNA phage M13mp2 exposed to sunlight: predominance of G-to-C transversion.

Sunlight is regarded to be a cause of skin cancer, though the mechanisms underlying the causation are still unclear. The genotoxic effects of sunlight are believed to be induced by pyrimidine photoproducts produced by the action of the UV portion of sunlight. However, it is not clear whether these pyrimidine modifications are the sole sources for the mutations. In the present study, we have analyzed the mutagenic potential of sunlight on the lacZ alpha region of single-stranded DNA phage M13mp2 using an SOS-deficient recA- strain and an SOS-induced rec+ strain of Escherichia coli as hosts. Exposure to sunlight caused mutations; approximately 10-fold increases in the mutation frequency were observed with the use of both hosts. When SOS functions were induced in the host CSH50, the mutation frequencies increased another 10-fold over those obtained with the host lacking the SOS functions. DNA sequences of the mutants were analyzed by automated DNA sequencers. Sequence changes were identified in 53 mutants from the mutant DNAs obtained using NR9099 as host and in 78 mutant samples obtained using UV-treated CSH50. Most of the mutations were transversions of guanine, either G to C or G to T. Furthermore, 59% of the identified sequence changes in the SOS- host and 40% of those in the SOS-induced host were G-to-C transversions. These transversions may be caused by unidentified guanine damages or by the effects of damage at pyrimidines distal from guanines to be mutated.