Infusional therapy with alkylating agents

Newborns with chronic problems needing continuous and special care even after discharge are not very frequent but represent a challenge for the caring team. The discharge program of the Neonatal Care ward of Trento hospital includes several steps: discharge meetings of teh neonatologist and the nurse responsible for the child, the head nurse, the psychologist and, when possible, the social worker; a training program for the parents; the coordination of communications and interventions of the home-care nurses and a detailed post-discharge planning. From 1995 a home-hospital program, as an alternative to the hospital admission was started. To describe how the team functions and stress the need of a close integration among the team members, the case of Ahmed is presented. This case faced the team with several challenges, because of the lack of parent's knowledge of the italian language and of the severity of the child's problems. Every care plan is developed building on newborn's needs and patients' resources. Data on the patients-problems dealt with from 1991 to 1995 and the interventions and resources needed are presented.     

Cocarcinogenic and tumor-promoting agents in tobacco carcinogenesis

A series of 21 tobacco smoke components and related compounds werere applied to mouse skin (50 female ICR/Ha Swiss mice/group) three times weekly with a low dose (5 mug/application) of benzo[a]pyrene (B[a]P). The test compounds were of five classes: aliphatic hydrocarbons, aromatic hydrocarbons, phenols, and long-chain acids and alcohols. The following compounds enhanced remarkably the carcinogenicity of B[a]P: catechol, pyrogallol, decane, undecane, pyrene, benzo[e]pyrene, and fluoranthene. The following compounds inhibited B[a]P carcinogenicity completely: esculin, quercetin, squalene, and oleic acid. Phenol, eugenol, resorcinol, hydroquinone, hexadecane, and limonene partially inhibited B[a]P carcinogenicity. Six of the 21 compounds were also tested as tumor promoters im two-stage carcinogenesis. No direct correlation existed between tumor-promoting activity and cocarcinogenic activity. The cocarcinogens pyrogallol and catechol did not show tumor-promoting activity. Decane, tetradecane, anthralin, and phorbol myristate acetate showed both types of activity. Structure-activity relationships and possible modes of action were described.     

Comparative study of three antineoplastic alkylating agents on DNA

The influence of three alkylating anticancer preparations phosphamide, sarcolysine, cyclophosphane on content of the 5-methylcytosine and parameters of the melting DNA of the liver healthy animals and tumor sarcoma 45 was investigated. It was shown, that among the investigated preparations cyclophosphane has stronger anticancer influence and comparatively weaker side effect on DNA liver. We came to the conclusion that it is preferable to use this preparation.     

Molecular basis for thymidine modulation of the efficacy and toxicity of alkylating agents

The antitumor activity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in mice previously was shown to be markedly enhanced by co-administration of thymidine. We have examined the cellular mechanisms underlying the augmentation effect of thymidine. It was found that thymidine did not increase the cytotoxicity of BCNU for B16/F10 melanoma or L1210 leukemia cells in vitro. Instead, thymidine appeared to augment the activity of tumor-specific cytotoxic T-cells in tumor-bearing mice, which specifically rejected a secondary challenge with the B16/F10 tumor. Thus, development of an antitumor immune response is facilitated by thymidine in BCNU-induced immunosuppressed mice. These preclinical studies suggested that combination therapy with alkylating agents and thymidine may be a more efficacious and less toxic anticancer therapy. The potential efficacy of the sequential administration of dacarbazine (DTIC), BCNU, and thymidine in patients with advanced malignant melanoma was investigated. As predicted from animal studies, sequential administration of DTIC, BCNU, and thymidine is a relatively nontoxic therapy for metastatic melanoma. This treatment induced durable responses in up to 35% of patients, and hence is superior to many commonly used toxic combination chemotherapies. The mechanism of action, although not well characterized, is thought to be mediated through protection of the cellular immune process, as well as organ function, from alkylating agent toxicity through modulation of DNA repair enzymes such as O(6)-alkylguanine-DNA alkyltransferase in normal tissue. Thus, thymidine is a biomodulator, which not only protects patients from hematologic, pulmonary, and hepatic toxicities associated with DTIC and BCNU chemotherapy, but also potentiates therapeutic efficacy.     

Studies on liver chromatin RNA from rats treated with alkylating agents

We have examined firstly some properties of rat liver chromatin RNA and nuclear sap RNA and secondly the incorporation of [3H]orotic acid into the RNA in vivo in control rats and in rats treated with the alkylating agents, N,N-dimethylnitrosamine or methyl methane sulphonate. Half or more of the nuclear RNA is associated with the chromatin and consists mainly of two species: one is labelled and probably comprises "nascent" RNA, and the other is unlabelled and of lower molecular weight. Neither species is attributable to cytoplasmic contamination. Studies with added polylysine with RNAase A and with DNAase I suggest that both species are ironically bound to protein and that the labelled species is not associated with the part of the chromatin DNA most readily degraded by DNAase I. After dimethylnitrosamine treatment, the amount of unlabelled RNA remains constant but the amount of labelled RNA increases after a low dose, and decreases after a high dose. After methyl methane sulphonate treatment, no change occurs in either species. These results can be explained by changes in extent of association of the DNA and protein within the chromatin complex.     

Influence of DNA repair by ada and ogt alkyltransferases on the mutational specificity of alkylating agents

We investigated the influence of the alkyltransferases (ATases) encoded by the ada and ogt genes of Escherichia coli on the mutational specificity of alkylating agents. A new mutational assay for selection of supF- mutations in shuttle-vector plasmids was used. Treating plasmid-bearing bacteria with N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), and ethyl methanesulfonate (EMS) dramatically increased the mutation frequency (from 33-fold to 789-fold). The vast majority of mutations (89-100%) were G:C-->A:T transitions. This type of mutation increased in ada- (MNU) or ogt- (ENU) bacteria, suggesting that repair of O6-methylguanine by ada ATase and repair of O6-ethylguanine by ogt ATase contribute mainly to the decrease in G:C-->A:T transitions. The analysis of neighboring base sequences revealed an overabundance of G:C-->A:T transitions at 5'-GG sequences. The 5'-PuG bias increased in ATase-defective cells, suggesting that these sequences were not refractory to repair. G:C-->A:T transitions occurred preferentially in the untranscribed strand after in vivo exposure. That this strand specificity was detected even in bacteria devoid of ATase activity (ada- ogt-) and not after in vitro mutagenesis suggests a bias for damage induction rather than for DNA repair. Highly significant differences were found between the in vivo and in vitro incidences of G:C-->A:T substitutions at the two major hotspots, positions 123 (5'-GGG-3'; antisense strand) and 168 (5'-GGA-3'; sense strand). These results are explained by differences in the probability of formation of stem-loop structures in vivo and in vitro.     

Retinyl substituted-benzyl ethers. Inhibition of mammary carcinogenesis by retinyl 3,4,5-trimethoxybenzyl ether (RTMBE)

Recently, we reported that retinyl 2-propynyl ether (RPE) inhibits MNU-induced mammary cancer in rats and is less toxic than RME and retinyl acetate. The preparation and biological investigations of retinyl ethers have now been extended to retinyl substituted-benzyl ethers, some of which bind to cellular retinol-binding protein. In long-term (160-180 days) experiments, retinyl 3,4,5-trimethoxybenzyl ether (RTMBE) has been shown to be active against MNU-induced mammary cancer in Sprague-Dawley rats. In effectiveness, RTMBE is comparable, at least, to retinyl acetate; but, unlike retinyl acetate, RTMBE is comparatively non-toxic to rats and mice, is not converted enzymatically to retinol, and does not cause significant increases in retinyl palmitate concentrations in the liver. RTMBE reaches high concentrations in mammary tissue. Two of the four RTMBE congeners that were evaluated in 90 day studies were moderately effective in inhibiting mammary carcinogenesis.     

DNA strand breaks and repair synthesis in Yoshida sarcoma: cells with differential sensitivities to bi-functional alkylating agents and UV light

The induction of DNA-strand breaks and repair synthesis has been examined in cultured Yoshida sarcoma cell lines sensitive (YS) and resistant (YR) to methylene dimethanesulphonate (MDMS). Using an alkaline DNA unwinding-hydroxylapatite technique, we were able to detect breaks in DNA immediately after MDMS treatment and at similar levels in both YS and YR cells. MDMS treatment and post-treatment incubation in the presence of 1-beta-D-arabino-furanosylcytosine (araC) lead to a large increase in the numbers of breaks when compared with MDMS treatment alone which indicated that many of the DNA-strand breaks seen after MDMS treatment were intermediates in excision repair. The magnitude of break incidence with the araC treatment was again equal in YS and YR cells indicating that these 2 lines made enzymic incisions next to MDMS-induced lesions with equal capacities. During incubation following MDMS treatment, the levels of DNA-strand breaks in YR cells were found to decrease more rapidly than in YS cells. Parallel DNA-repair synthesis estimations, using BND-cellulose chromatography, revealed that the increased rate of decline in breaks in YR cells was accompanied by an increase in repair-synthesis activity compared to YS cells. This was interpreted as indicating that an intermediate step in an excision-repair pathway for MDMS-induced lesions was relatively deficient in YS compared to YR cells. A similar difference in the rates of decline of DNA-strand breaks between YS and YR cells was also observed following treatment with UV light to which MDMS-resistant YR cells also display cross-resistance. However, no such difference was detected following treatment with the monofunctional alkylating agent, methyl methanesulphonate, to which YS and YR cells are equally sensitive. These results suggest that resistance to MDMS in the YR cell line is achieved by an increased efficiency in the gap-sealing component of the excision-repair process.     

Rapidly cycled courses of high-dose alkylating agents supported by filgrastim and peripheral blood progenitor cells in patients with metastatic breast cancer

Our purpose was to determine the feasibility of a regimen of multiple, rapidly cycled courses of high-dose alkylating agents, including paired courses of escalating doses of thiotepa, supported by peripheral blood progenitor cells and filgrastim, in patients with responding stage IV breast cancer. The regimen consisted of two courses of cyclophosphamide (3.0 g/m2/course) followed by two courses of thiotepa (500-700 mg/m2/course). All courses were supported by filgrastim. Leukaphereses were performed after each cyclophosphamide course to harvest peripheral blood progenitors (PBPs) for use as rescue following thiotepa administration. The planned interval for all courses was 14 days. Forty-two patients were enrolled. Thirty-eight received all four courses, and four did not receive the second thiotepa cycle due to poor PBP mobilization. The maximum dose of thiotepa that was administered was 700 mg/m2 x 2. At this dose, one patient developed encephalopathy, which resolved over several weeks. The median number of days to an absolute neutrophil count of 0.5 x 10(9)/liter after PBP reinfusion for cycles 1 and 2 of thiotepa were 9 (range, 7-16) and 9 (range, 8-13) days, respectively. The corresponding values for platelet recovery to >20 x 10(9)/liter were 11 (range, 8-39) and 12 (range, 10-28) days, respectively. There were no treatment-related deaths. Hospitalization was required following 28 of 84 cyclophosphamide courses and 76 of 80 thiotepa courses. Four patients developed grade III-IV mucositis. The median interval between courses of treatment was 15 (range, 13-29) days. Of 19 patients who entered the protocol with measurable disease in partial response from prior therapy, 8 (42%) achieved complete response following the high-dose therapy. Nine (21%) of 42 remain progression free at a median follow-up of 28 (range, 20-32) months. Therefore, we concluded that the administration of multiple, rapidly cycled courses of high-dose alkylating agents is feasible.     

可溶性聚醚醚酮改性环氧树脂的研究

采用热熔法制备了一系列可溶性聚醚醚酮(s-PEEK)改性环氧树脂(EP),并与普通聚醚醚酮(PEEK)改性环氧体系进行比较,探讨了聚醚醚酮类型、用量对改性树脂固化体系的凝胶时间、冲击强度、弯曲性能和断裂形貌的影响,并对含s-PEEK树脂体系的玻璃化转变温度(Tg)和热稳定性进行了分析.结果表明,s-PEEK和PEEK可在提高环氧体系冲击性能的同时,提高材料的弯曲性能、玻璃化温度和热稳定性;当m(s-PEEK):m(E-51)和m(PEEK):m(E-51)均为5:100时,冲击强度达到42.6和46.6 kJ/m2,分别比未改性的环氧体系提高69.1%和85.6%;m(s-PEEK):m(E-51)=25:100时,Tg=179.1℃,比未改性环氧树脂提高20℃左右;且含s-PEEK的体系是均相体系,含s-PEEK的固化物是颗粒增强体系.     

The human immunodeficiency virus type 1 long terminal repeat is activated by monofunctional and bifunctional DNA alkylating agents in human lymphocytes

The activation of the human immunodeficiency virus, type 1 (HIV-1) by the DNA alkylating agents ethyl methanesulfonate, methyl methanesulfonate, and mitomycin C was observed in human B lymphocytes transiently transfected with plasmids in which the HIV-1 long terminal repeat (LTR) directed the expression of the bacterial chloramphenicol acetyltransferase gene. Deletion of the two NF-kappa B-binding sites of LTR abolished the HIV-1 activation induced by the three mutagens, while deletion of the three Sp1-binding sites slightly reduced it. Electrophoretic mobility shift assays revealed an increased binding to the kappa B sites of HIV-1 LTR in the nuclear extracts of human B lymphocytes upon mutagen treatment, while binding to Sp1 sites was unaffected. The TAR region was also involved in the mutagen-mediated activation of HIV-1 LTR inasmuch as a small deletion in the TAR sequence (nucleotides +34 to +37) greatly decreased the induction of HIV-1 expression. Moreover, an enhanced binding activity to the TAR DNA sequence (nucleotides +24 to +47) was observed in nuclear extracts of mutagen-treated lymphocytes. Thus, both the enhancer and the 5'-untranslated region of HIV-1 functionally cooperate in the mutagen-mediated induction of HIV-1 expression.     

Effect of folate deficiency on mutations at the hprt locus in Chinese hamster ovary cells exposed to monofunctional alkylating agents

Multiplex PCR amplification of hprt exons from 113 Chinese hamster ovary cell clones selected for resistance to 6-thioguanine was performed to investigate the molecular basis for the synergistic mutagenic effects of nutritional folic acid deficiency and alkylating agents. In cells treated with ethyl methanesulfonate, intragenic deletions were detected in 9 of 46 (19.6%) clones derived from folate-deficient cells, but in none of 16 mutants grown in folate-replete medium. The number of deletions found in mutants generated by N-nitroso-N-ethylurea was low in both folate-deficient (1 of 25; 4%) and folate-replete (1 of 26; 3.8%) cells. Correction of folate deficiency may decrease the frequency of intragenic deletions caused by some alkylating agents.     

Apoptosis and carcinogenesis

Many tumours are characterised by increased levels of apoptosis. This observation establishes significance for this process in tumour development, but it does little to elucidate the nature of this role, nor does it yield information relevant to the early stages of carcinogenesis. To gain a better understanding of the importance of apoptosis, it has been necessary to create a number of transgenic model systems wherein the apoptotic response has been modified. Using this strategy, a number of genetic lesions have been identified which affect both the apoptotic pathway and predisposition to malignancy. These lesions can operate either directly, by blocking the induction of apoptosis; or indirectly, by increasing the selective pressure for further genetic change. The consequent deregulation of growth control and increase in mutation burden represent two key steps in carcinogenesis, underlining the pivotal role played in tumour suppression by the normal induction of apoptosis.     

Transplacental and transgenerational carcinogenesis

Effects of exposure to carcinogens at early stages of ontogenesis are considered. An increased cancer risk due to prenatal exposure may be related to: 1) exposure of the fetus during pregnancy to chemicals able to cross the placental barrier or to radiation; 2) exposure to a chemical or radiation of the parents or one parent prior to conception. In transplacental carcinogenesis, the effects observed after birth are a consequence of a direct interaction of the carcinogen with somatic cells of the fetus. DES and radiation were shown to increase cancer risk in humans following exposure during pregnancy, while in experimental animals a large variety of chemicals of quite different structure (including the widely used therapeutic agent cisplatin) were demonstrated to induce tumors in the progeny after administration during pregnancy. The experimental multigeneration effect of carcinogens is manifested in an increased incidence of tumors in several generations of untreated descendants of: a) females exposed to carcinogen during pregnancy; b) males exposed to carcinogen prior to mating with untreated females. The inherited change may be an initiating event revealed by the exposure during post-natal life to a promoting agent. In humans deleterious information inherited through the germ cells (occurring either following a spontaneous error in DNA replication and repair or as a consequence of a chemical or physical agent) can increase the risk of developing cancer in certain individuals by several orders of magnitude (retinoblastoma, familial polyposis of the colon and some others). The multigeneration transmission of carcinogenic risk is also demonstrated by cancer prone families that are probably more frequent than originally thought, with a risk that is one order of magnitude higher than in general population. Familial clusterings of cancer may also indicate germline mutations in one or more genes. Thus the inherited predisposition to cancer that is observed today may, at least in part, be explained by the exposure to environmental noxious agents in previous generation(s). Since humans are exposed throughout life to many environmental agents, either carcinogenic or capable to enhance the progression of cancer, an understanding of the contribution of prenatal exposure to carcinogens could improve the efficacy of prevention.     

Genomic imprinting and carcinogenesis

The solution structure of a synthetic ET(B) selective agonist, ET-1[Cys(Acm)(1,15), Ala3, Leu7, dAsp8, Aib11] has been solved by 1H NMR and molecular modelling studies. Such solution structures of linear modified peptides in aqueous methanol are being used in an ongoing program of research designed to assist in an understanding of the basic structural requirements for the biological activity of vasoconstrictors. The resulting structure of this peptide is characterised by an alpha-helical conformation between residues Leu6-His16 and by N- and C-termini which assume no defined conformation. A knowledge of the solution structures of this and related peptides, which are ET(B) selective agonists, are proving to be important in the understanding of how they interact with the ET(B) receptor.     

Biodegradation of the gasoline oxygenates methyl tert-butyl ether, ethyl tert-butyl ether, and tert-amyl methyl ether by propane-oxidizing bacteria

Several propane-oxidizing bacteria were tested for their ability to degrade gasoline oxygenates, including methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME). Both a laboratory strain and natural isolates were able to degrade each compound after growth on propane. When propane-grown strain ENV425 was incubated with 20 mg of uniformly labeled [14C]MTBE per liter, the strain converted > 60% of the added MTBE to 14CO2 in < 30 h. The initial oxidation of MTBE and ETBE resulted in the production of nearly stoichiometric amounts of tert-butyl alcohol (TBA), while the initial oxidation of TAME resulted in the production of tert-amyl alcohol. The methoxy methyl group of MTBE was oxidized to formaldehyde and ultimately to CO2. TBA was further oxidized to 2-methyl-2-hydroxy-1-propanol and then 2-hydroxy isobutyric acid; however, neither of these degradation products was an effective growth substrate for the propane oxidizers. Analysis of cell extracts of ENV425 and experiments with enzyme inhibitors implicated a soluble P-450 enzyme in the oxidation of both MTBE and TBA. MTBE was oxidized to TBA by camphor-grown Pseudomonas putida CAM, which produces the well-characterized P-450cam, but not by Rhodococcus rhodochrous 116, which produces two P-450 enzymes. Rates of MTBE degradation by propane-oxidizing strains ranged from 3.9 to 9.2 nmol/min/mg of cell protein at 28 degrees C, whereas TBA was oxidized at a rate of only 1.8 to 2.4 nmol/min/mg of cell protein at the same temperature.     

Ultraviolet radiation and skin carcinogenesis

A case report of boutonneuse fever with pulmonary complications in a patient with non-Hodgkin's lymphoma (NHL) is described. The patient was hospitalized for persistent hypertermia and marked dyspnea, with radiographic findings of bilateral involvement of the lungs. The confirmation of the diagnosis was obtained by means of serum analyses (Weil-Felix serodiagnosis and IFA); the patient responded to doxycycline with progressive improvement of her general health condition. In this case the occurrence of a NHL could justify the lower reactivity and the facilitated diffusion of rickettsiosis in the patient.