Volume-controlled versus short drainage after inguinofemoral lymphadenectomy in vulvar cancer patients: A Dutch nationwide prospective study.

OBJECTIVE: Inguinofemoral lymphadenectomy for patients with vulvar squamous cell carcinoma is associated with a high incidence of postoperative wound complications, which may be influenced by inguinal drain management. The aim of this nationwide prospective study (MAMBO: Morbidity And Measurement of the BOdy) was to assess the feasibility and the incidence of complications after volume-controlled versus short drainage. METHODS: The MAMBO study consisted of two observational studies in all eight oncology centers in the Netherlands, conducted between 2012 and 2016. In the first study, the drain was removed when the production was <30ml/24h, except in the first 48h, and after a maximum of 28days (MAMBO-IA). In the second study, the drain was removed five days postoperatively regardless of production (MAMBO-IB). We assessed the complications within eight weeks after surgery using logistic regression to compare the incidence of one or more complications between the two drainage protocols, adjusting for possible confounders. RESULTS: We included 77 patients (139 groins) for volume-controlled drainage and 64 patients (112 groins) for short drainage. Volume-controlled drainage was associated with significant less lymphocele formation. Moreover, we found no difference in wound infection or primary wound breakdown. The estimated incidence of one or more complications was 46% per groin after volume-controlled drainage versus 75% after short drainage, (RD 29% (95% CI 8, 49) p=0.006). CONCLUSIONS: This prospective study shows that volume-controlled drainage is associated with significantly less complications compared to short drainage. We therefore recommend volume-controlled drainage after inguinofemoral lymphadenectomy in patients with vulvar squamous cell carcinoma.

Interpreting biomarker data from the COPHES/DEMOCOPHES twin projects: Using external exposure data to understand biomarker differences among countries.

In 2011 and 2012, the COPHES/DEMOCOPHES twin projects performed the first ever harmonized human biomonitoring survey in 17 European countries. In more than 1800 mother-child pairs, individual lifestyle data were collected and cadmium, cotinine and certain phthalate metabolites were measured in urine. Total mercury was determined in hair samples. While the main goal of the COPHES/DEMOCOPHES twin projects was to develop and test harmonized protocols and procedures, the goal of the current paper is to investigate whether the observed differences in biomarker values among the countries implementing DEMOCOPHES can be interpreted using information from external databases on environmental quality and lifestyle. In general, 13 countries having implemented DEMOCOPHES provided high-quality data from external sources that were relevant for interpretation purposes. However, some data were not available for reporting or were not in line with predefined specifications. Therefore, only part of the external information could be included in the statistical analyses. Nonetheless, there was a highly significant correlation between national levels of fish consumption and mercury in hair, the strength of antismoking legislation was significantly related to urinary cotinine levels, and we were able to show indications that also urinary cadmium levels were associated with environmental quality and food quality. These results again show the potential of biomonitoring data to provide added value for (the evaluation of) evidence-informed policy making.

First assessment of population exposure to perfluorinated compounds in Flanders, Belgium.

With the objective to evaluate exposure of the population in Flanders (Belgium) to perfluorinated compounds (PFCs), we measured perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) in settled dust in homes and offices, in a selection of food items from local origin, in drinking-water and in human serum. We complemented the data with results from a literature survey. Based on this dataset we calculated intake by children and adults from food, drinking-water, settled dust and soil, and air. Dietary exposure dominated overall intake. For adults, average dietary intake equalled 24.2 (P95 40.9) ng PFOS kg(-1) d(-1) and 6.1 (P95 9.6) ng PFOA kg(-1)d(-1), whereas for children the dietary intake was about 3 times higher. Predicted intake is high when compared to assessments in other countries, and to serum levels from Flanders, but comparable to the intakes published by The European Food Safety Authority (EFSA) in 2008. Intake of PFOS and PFOA remained below the Tolerable Daily Intake.

Predicting blood lead levels from current and past environmental data in Europe.

The present case study on lead in Europe illustrates the use of the Integrated Monitoring Framework Strategy to assess the health outcome of environmental pollution by evaluating the associations between lead in various environmental compartments (air, soil, dust, drinking water and diet) and lead concentrations in blood (B-Pb) for various age-related sub-populations. The case study was aimed to investigate whether environmental, exposure and biomonitoring data at general population level, covering all EU member states, could be integrated. Although blood lead has been monitored extensively in Europe, consistent datasets are not yet available. Data diverge with regard to objectives, regional scale, sampling years, gender, age groups and sample size. Significant correlations were found between B-Pb and the concentrations of Pb in air and diet. The significant decrease of the Pb in air over time from 0.31 µg/m(3) (P95: 0.94; n=98) prior to 1990 to 0.045 µg/m(3) (P95: 0.11; n=256) in 2007 (latest observations included) (Δ=-85%) corresponds to a decline in B-Pb by 48% and 57% in adult women and adult men, respectively. For pre-school children a more shallow decline in B-Pb of 16% was calculated over the same period. Similarly, the reduction in Pb-dietary intake from on average 68.7 µg/d (P95: 161.6; n=19) in 1978 to 35.7 µg/d (P95: 82.3; n=33) in the years post 2000 (Δ=-48%) is paralleled by a decline in B-Pb of 32, 33 and 19% in adult women, primary- and pre-school children, respectively. Insufficient data exist for other age groups to calculate statistically significant correlations. Although regression models have been derived to predict B-Pb for different sub-populations in Europe based on Pb concentrations in air and soil as well as dietary intake, it is concluded that the available data are insufficient to accurately predict actual and future simultaneous exposure to Pb from various environmental compartments, and as a consequence the health impact of Pb for various target populations at EU scale. At least due to data availability, air Pb remains the best predictor of B-Pb in the population. However, lead emission sources have largely been reduced and inhalation of lead in air is not causal to B-Pb levels. Therefore, there is a need of adequate data for Pb in soil and house dust, and in diet and drinking water as these are causal exposure sources with a longer Pb half-life than air. An extended and more harmonized surveillance system monitoring B-Pb, especially in children, is urgently required in order to identify, quantify and reduce still remaining sources of Pb exposure.

The use of biomarkers for risk assessment: Reporting from the INTARESE/ENVIRISK Workshop in Prague.

This publication is a report on the workshop "The use of biomarkers for risk assessment" which took place in November 2007 in Prague, the Czech Republic. The main aim of the workshop was to bring together a broad international audience with a particular interest in the development and application of human biomonitoring (HBM) and biomarkers for environmental health research, and to provide a state-of-the art overview of the potential values and pitfalls of biomarkers in risk assessment. Throughout the presentations and the subsequent discussions, it was shown that human biomonitoring is a highly plastic and versatile tool for the unraveling of the link between contaminants in the environment and potentially associated health effects in the general population. Although it offers a means to integrate exposure through different environmental compartments, to integrate exposure over time, to include individual risk factors and genetic susceptibility, exposure biomarkers would greatly benefit from standardized, accurate and sensitive detection methods and toxicokinetic data. Effect biomarkers on the other hand need to be put into their relevant public health perspective, and well validated, mechanistically sound dose-response relationships are essential. New developments, such as in vitro assays and "-omics", may drastically improve our knowledge on the causal mechanisms behind environmental health associations and will allow for a more informed linkage of toxicological and epidemiological reality.

Availability and comparability of human biomonitoring data across Europe: a case-study on blood-lead levels.

Recently, it has become clear that the complexity of environmental health issues requires an approach that takes into account the complexities, interdependencies and uncertainties of the real world. An urgent issue that has surfaced is the need for accurate tools to better describe exposure characterization to environmental chemicals. By including human biomonitoring (HBM) data, a greater precision in exposure and associated risk estimates and more accurate dose-response relationships may be achieved. A restricting issue still is the availability of reliable and comparable HBM data. The aim of the current study was twofold: (1) to find out whether it is practically feasible to collect raw, individual HBM data across Europe; and (2) to evaluate the comparability and use of these HBM data for environmental health impact assessment at a European scale. Blood-lead (B-Pb) was selected as the chemical of choice because of its long history as an environmental pollutant in HBM programs and its known public health relevance. Through literature search and identification of HBM experts across Europe, HBM programs that measured B-Pb were identified and asked to share individual data on age, gender and B-Pb levels. Following this request, more than 20,000 individual data points from 8 European countries were collected. Analysing these data made clear that it is difficult to use disparate data collections because of the inherent variability with respect to the gender and age of participants and calendar-years sampled. When these confounders were taken however, there was no additional variability in B-Pb distributions among different countries. It was concluded that while it is possible to collect HBM data from different sources across Europe, the need to get data from comparable (sub-)populations is essential for appropriate use and interpretation of HBM data for environmental health impact assessment.

Translating biomonitoring data into risk management and policy implementation options for a European Network on Human Biomonitoring.

BACKGROUND: The "European Environment & Health Action Plan 2004-2010" originates from the concern of the European Commission on the well-being of individuals and the general population. Through this plan, the Commission has set the objectives to improve the information chain for a better understanding of the link between sources of pollution and health effects, to better identify existing knowledge gaps, and improve policy making and communication strategies. Human biomonitoring (HBM) has been included as one of the tools to achieve these objectives. As HBM directly measures the amount of a chemical substance in a person's body, taking into account often poorly understood processes such as bioaccumulation, excretion, metabolism and the integrative uptake variability through different exposure pathways, HBM data are much more relevant for risk assessment than extrapolations from chemical concentrations in soil, air, and water alone. However, HBM primarily is a stepping stone between environmental and health data, and the final aim should be an integrated and holistic systematic risk assessment paradigm where HBM serves as a pivotal point between environment and health, on the one hand leaning on environmental data to provide detailed information on the sources and pathways of pollutants that enter the human body, and on the other hand clarifying new and existing hypotheses on the relationship between environmental pollutants and the prevalence of diseases. With the large amount of data that is being gathered in the different national survey projects, and which is expected to become available in Europe in the near future through the expected European Pilot Project on HBM, a framework to optimize data interpretation from such survey projects may greatly enhance the usefulness of HBM data for risk managers and policy makers. RESULTS: This paper outlines an hierarchic approach, based on the stepwise formulation of 4 subsequent steps, that will eventually lead to the formulation of a variety of policy relevant risk reduction options. CONCLUSION: Although the usefulness of this approach still needs to be tested, and potential fine-tuning of the procedure may be necessary, approaching the policy implications of HBM in an objective framework will prove to be essential.

Prokaryotic gene profiling assays to detect sediment toxicity: evaluating the ecotoxicological relevance of a cell-based assay.

Despite their complexity, ecotoxicological measurements using higher level responses remain a major tool in the assessment of ecosystem integrity. Nevertheless, the past decade saw an increasing number of cell based testing systems have found widespread application in ecotoxicology. One such test is bacterial bioreporters carrying a stress sensitive promoter fused to an easily detectable reporter gene. In the presence of a specific toxic stress,the expression cassette is switched on and the reporter gene is produced. This study evaluated the use of 14 different Escherichia coli bioreporter strains sensitive to different types of toxicity in the assessment of the ecological status of a small river basin in Flanders, Belgium. The river is fed at two geographically separate locations by two distinct and well-described effluents, one from a household sewage treatment facility and one from the discharge of the wastewater treatment facility of a large chemical plant. The results of the bacterial gene profiling assay were related to active biomonitoring results obtained through higher-level responses of caged Dreissena polymorpha, Chironomus riparius, and Cyprinus carpio deployed at the locations sampled for the bacterial assay. The results of the gene induction assay and the active biomonitoring data correlated well and corresponded to the flow dilution data, which is used here as a surrogate forthe chemical pollution gradient present in the river basin.

Identifying opportunities and gaps for establishing an integrated EDR-triad at a European level.

It is the explicit objective of the ESBIO project (Expert team to Support BIOmonitoring in Europe) to develop a harmonized and integrated human biomonitoring (HBM) framework within the EU, and to elaborate how HBM can be integrated most efficiently with environmental monitoring and registered health data. Work package 3 of the ESBIO projects aims at developing scenarios for linking biomonitoring data to available data on environmental exposure and population health. Although it is recognized that there is a wide variety of data available, it is often difficult to integrate these different data layers because of differences in database structures, geographical detail and spatial distribution, or most importantly because the data simply were not meant to be interpreted in the context of integrated human risk assessment. This paper briefly explores the available information on Europe-wide environmental quality and health data that could be used in cooperation with HBM. Because ESBIO focuses on the whole of Europe, but also needs opportunities for further refinement on a more detailed local scale, the applicability of geographical information systems (GIS) in environmental health, HBM and human health assessments were highlighted. It was concluded that there is an abundance of information on the presence and behavior of pollutants in the environment for some compartments (e.g., ambient air), while for other compartments, measurements are more difficult to gather, and/or no clearly defined geographically explicit networks appear to be in place.

Oral estradiol decreases plasma homocysteine, vitamin B6, and albumin in postmenopausal women but does not change the whole-body homocysteine remethylation and transmethylation flux.

Estrogens, both endogenous and exogenous, lower the fasting levels of the independent risk factor for cardiovascular disease homocysteine. The mechanism behind this observation remains unclear. In a randomized, placebo-controlled, double-blind study, 25 postmenopausal women with a screening homocysteine concentration above 10 micromol/liter were included. We investigated the influence on homocysteine levels of a 3-month treatment with a daily oral dose of 4 mg 17beta-estradiol (ET) or 4 mg ET combined with 10 mg dydrogesterone (EPT); the comparison group received placebo treatment. We performed primed continuous infusions of L-[2H3-methyl-13C]methionine to assess steady-state flux rates of transmethylation, remethylation, and transsulfuration. Homocysteine concentration relationships with S-adenosylmethionine, S-adenosylhomocysteine, creatinine, albumin, vitamins B6 and B12, and folate status were determined as well. The mean change from baseline in homocysteine concentration by both treatments compared with placebo (ET, -13%; EPT, -10%) was accompanied by a decrease in the concentration of vitamin B6 (ET, -25%; EPT, -38%) and albumin (ET, -7%; EPT, -11%). No significant changes in flux rates were observed. In a .multiltivariate analysis, changes in homocysteine concentration were related to changes in albumin concentration. No relation to other variables was observed. We conclude that the ET- and EPT-induced homocysteine changes in this study were not accompanied by a significant change in methionine-homocysteine flux rates and hypothesize that an estrogen-induced lowering of homocysteine levels is primarily part of a change in albumin metabolism.

Hormone replacement influences homocysteine levels in the methionine-loading test: a randomized placebo controlled trial in postmenopausal women.

OBJECTIVE: To investigate the mechanism by which exogenous oestrogen influences the homocysteine metabolism in postmenopausal women. STUDY DESIGN: A randomized placebo controlled trial in which a methionine-loading test was performed, in 25 healthy postmenopausal women, before and after a 12-week oral treatment with placebo or daily 4 mg 17beta-estradiol with (HRT) or without (ERT) 10 mg dydrogesterone. Fasting and post-load homocysteine as well as Vitamins B(6), B(12) and folate were determined. RESULTS: In both treatment groups a significant 12% decrease in fasting homocysteine was observed. This decrease was accompanied by a post-load homocysteine increase of more than 20%. Vitamin B(6) values were decreased by more than 25%. CONCLUSION: The hormone therapy induced lowering of fasting homocysteine and Vitamin B(6) levels and an increase in post-load homocysteine, supporting the hypothesis that homocysteine-methionine metabolism is modulated by hormone therapy in postmenopausal women.

In situ and laboratory bioassays to evaluate the impact of effluent discharges on receiving aquatic ecosystems.

Effluents are a main source of direct and often continuous input of pollutants into aquatic ecosystems with long-term implications on ecosystem functioning. Therefore, the study of the effects of effluent exposure on organisms, populations or communities within the framework of impact assessment has a high ecological relevance. The aim of this study was to assess the toxicological impact of two effluents, one household wastewater treatment effluent (Effluent 1) and one industrial effluent (Effluent 2), on the receiving aquatic ecosystem using two test species under both in situ and laboratory conditions. Zebra mussel (Dreissena polymorpha) and common carp (Cyprinus carpio) were exposed under laboratory conditions in an online monitoring flow-through system (receiving different concentrations of Effluent 2) and under in situ conditions along the pollution gradient established by these two effluent discharges. Bioassays focussed on growth and condition related endpoints (i.e. condition, growth, lipid budget), since these are key functional processes within organisms and populations. Under laboratory conditions, increasing concentrations of the industrial effluent (Effluent 2) had a negative effect on both zebra mussel and carp energy reserves and condition. Under in situ conditions, the same negative impact of Effluent 2 was observed for zebra mussels, while Effluent 1 had no apparent effect on exposed zebra mussels. Carp growth and condition, on the other hand, were significantly increased at the discharge sites of both effluents when compared to the reference site, probably due to differences in food availability. The results indicate that a combination of in situ and laboratory exposures can illustrate how ecological processes influence bioassay studies. The incorporation of indirect, ecological effects, like changes in food availability, provides considerable benefit in understanding and predicting effects of effluents on selected species under realistic exposure conditions.

An ecologically relevant exposure assessment for a polluted river using an integrated multivariate PLS approach.

A case study is presented where an integrated, ecologically relevant exposure assessment is presented for a polluted lowland river. Using partial least squares regression of latent structures (PLS), an analysis of the impact of two effluents on physico-chemical water quality measures, macroinvertebrate and diatom communities, and in situ bioassay responses with four different test species are combined into an integrative exposure assessment. Bioassays focussed on growth and condition related endpoints, because they are key functional processes of organisms and populations. Integrating these multiple lines of evidence, we were able to discriminate among the impact of both effluents, link changes in physico-chemical water quality with bioassay endpoints and ecological quality of the ecosystem, and address the importance of integrating all information into one exposure assessment framework. The bioassays under field conditions indicated that most endpoints measured are a reflection of ecological effects rather than pollution related effects, or at least a combination of both ecological and toxicological effects. Factors such as food availability clearly influenced the outcome of in situ bioassays and ecological information was essential to explain observed discrepancies when trying to extrapolate bioassay results from the laboratory to the field.

Cellular energy allocation in zebra mussels exposed along a pollution gradient: linking cellular effects to higher levels of biological organization.

Organisms exposed to suboptimal environments incur a cost of dealing with stress in terms of metabolic resources. The total amount of energy available for maintenance, growth and reproduction, based on the biochemical analysis of the energy budget, may provide a sensitive measure of stress in an organism. While the concept is clear, linking cellular or biochemical responses to the individual and population or community level remains difficult. The aim of this study was to validate, under field conditions, using cellular energy budgets [i.e. changes in glycogen-, lipid- and protein-content and mitochondrial electron transport system (ETS)] as an ecologically relevant measurement of stress by comparing these responses to physiological and organismal endpoints. Therefore, a 28-day in situ bioassay with zebra mussels (Dreissena polymorpha) was performed in an effluent-dominated stream. Five locations were selected along the pollution gradient and compared with a nearby (reference) site. Cellular Energy Allocation (CEA) served as a biomarker of cellular energetics, while Scope for Growth (SFG) indicated effects on a physiological level and Tissue Condition Index and wet tissue weight/dry tissue weight ratio were used as endpoints of organismal effects. Results indicated that energy budgets at a cellular level of biological organization provided the fastest and most sensitive response and energy budgets are a relevant currency to extrapolate cellular effects to higher levels of biological organization within the exposed mussels.

Estrogens, homocysteine, vasodilatation and menopause: basic mechanisms, interactions and clinical implications.

Estrogens influence the independent cardiovascular risk factor homocysteine as well as vasodilatation. Homocysteine alone also influences vasodilatation, indicating a relational triangle that seems important in interpreting the isolated effects of estrogens on homocysteine metabolism and vasoreactivity. This paper gives an overview of the current understanding regarding vasoreactivity, homocysteine metabolism and the role of estrogens. This is placed against the background of the clinical trials on the effect of postmenopausal hormone replacement therapy on homocysteine levels and addresses the importance of the interaction between homocysteine, estrogens and vasoreactivity.

A randomized placebo-controlled study of the effect of transdermal vs. oral estradiol with or without gestodene on homocysteine levels.

OBJECTIVE: To assess the effect of transdermal vs. oral administration of E2 on plasma homocysteine levels and to evaluate the impact of adding a progestogen to these regimens. DESIGN: Prospective, double-blind, double-dummy, placebo-controlled study. SETTING: Outpatient clinics in two university hospitals and two teaching hospitals in The Netherlands. PATIENT(S): One hundred fifty-two healthy hysterectomized postmenopausal women. INTERVENTION(S): Thirteen 28-day treatment cycles with placebo (n = 49); transdermal 17beta-E2, 50 microg (n = 33), oral E2, 1 mg (n = 37), or oral E2, 1 mg, plus gestodene, 25 microg (n = 33), followed by four cycles of placebo in each group. MAIN OUTCOME MEASURE(S): Fasting plasma total homocysteine concentrations at baseline and cycle 4, 13, and 17. RESULT(S): Mean (+/-SD) homocysteine concentrations in the oral E2 group decreased from baseline to cycle 4 (9.0 +/- 2.5 micromol/L vs. 8.2 +/- 2.0 micromol/L; mean change, -7.6%). Homocystine values in the oral E2 plus gestodene group did not change substantially from baseline to cycle 4 (8.9 +/- 1.6 micromol/L vs. 8.6 +/- 2.0 micromol/L; mean change, -4.4%). No significant changes were observed in the transdermal E2 group. After four washout cycles, the homocysteine concentration had returned to baseline values in all groups. CONCLUSION(S): Oral E2 therapy reduced the homocysteine concentration more than did therapy with transdermal E2 or oral E2 plus gestodene. This finding may indicate a role of liver metabolism and suggests that gestodene has a negative effect on these changes.

Active efflux of the 5-HT(1A) receptor agonist flesinoxan via P-glycoprotein at the blood-brain barrier.

The role of P-glycoprotein on the efflux of the 5-HT(1A) receptor agonist flesinoxan across the blood-brain barrier in vivo and in vitro was investigated. In vitro, the transport ratios (representing polarized transport) of flesinoxan (10 microg/ml) were 4.2 in the MDR1-transfected LLC-PK1 cell line, which could be inhibited by the Pgp modulators SDZ-PSC 833 and LY 335979 and 1.1 in the wild-type LLC-PK1 cell line after 4 h. Flesinoxan concentrations lower than 33 microg/ml were actively transported by Pgp, while at higher concentrations Pgp became saturated and transport in the MDR1-transfected cell line was comparable with the wild-type cell line. In the in vitro BBB co-culture model the transport ratio was 2.0 and was decreased to 1.0 in the presence of Pgp modulators. In vivo, the accumulation of flesinoxan in the brain at 3 h was much higher in the mdr1a(-/-) mice compared to mdr1a(+/+) mice (ratio 12.6 and 27.0 at dose levels of 3 mg/kg and 10 mg/kg respectively). In conclusion, both in vivo as well as in vitro results have demonstrated that Pgp is a limiting factor for the transport of the 5-HT(1A) receptor agonist flesinoxan into the CNS. This should be considered when its application in therapy is combined with other Pgp substrates.

Hormone replacement therapy and plasma homocysteine levels.

OBJECTIVE: To compare the effects of 4 and 12 weeks of combined estradiol-progestogen replacement with unopposed estradiol therapy on fasting plasma total homocysteine concentrations in healthy postmenopausal women. METHODS: In this prospective, 12-week study in healthy postmenopausal women, we randomly assigned 59 women to sequentially combined daily 2 mg estradiol (E2) plus either trimegestone 0.5 mg daily or dydrogesterone 10 mg daily (n = 28), or to unopposed daily 2 mg estradiol (n = 16), or to placebo (n = 15). RESULTS: Fasting plasma total homocysteine concentrations decreased by 9.4% in the combined estradiol-progestogen group and by 5.1% in the estradiol-only group, and they increased by 2.4% in the placebo group (analysis of covariance: combined hormone replacement therapy compared with placebo (P = .02); combined therapy compared with estradiol (P = .23); and estradiol compared with placebo (P = .26). Reductions were detectable after 4 weeks of combined estradiol-progestogen treatment. The data suggest an additional progestogen-related reduction in homocysteine levels of 0.7 micromol/L and 0.4 micromol/L after 4 and 12 weeks, respectively. Women with a baseline homocysteine concentration in the highest quartile had significantly greater reductions in homocysteine compared with women with an initial homocysteine value in the lowest quartile. CONCLUSION: Fasting total homocysteine concentrations were significantly reduced by combined estradiol-progestogen replacement. Women with high homocysteine levels at baseline benefit the most. The progestogens used in this study did not have an unfavorable effect on homocysteine metabolism.

[Hemisynthesis of antineoplastic conjugates with nitrogen-mustard proteins]. / Hémisynthèse de conjugués oncostatiques du type moutardes azotées-protéines.

The synthesis of new conjugates with inhibitory action on tumour growth is investigated by linking amino functions of proteins compounds (lysozyme and alpha s-casein) through an amide linkage at the carboxylic function of nitrogen mustards (chlorambucil and melphalan). The polychlorambucil amides of lysozyme and alpha s-casein derivatives prepared showed experimental antitumour activity when these conjugates were screened against the experimental P388 leukemia. In the case of the conjugates lysozyme-melphalan, an antitumour activity is observed when the amino function of the drug is combined with the carboxylic functions of the protein contrary to the situation of the free amino function of the drug described into the literature.

[Reaction of carbodiimide for the introduction of p-bis(2-chloroethyl)amino-L-phenylalanine to lysozyme]. / Réaction à la carbodiimide visant l'introduction de la p-bis-(2-chloroéthyl)amino-L-phénylalanine sur le lysozyme.

In the synthesis of new prodrugs with inhibitoring action of tumour growth, a new nitrogen mustard derivative was obtained, proceeding of the coupling between an egg-white lysozyme with an antitumor amine nucleophile, the methyl ester of p-bis-(2-chloroethyl)amino-L-phenylalanine (Melphalan), catalyzed by 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide (EDC), at pH 5.0 and room temperature. In that case, the mechanism for the modification isn't selective of Asp101 in lysozyme. As in cases of histamine and D-glucosamine [3], it is evident that Melphalan is one type of amine who doesn't cause a selective modification of Asp101 but causes somewhat random reaction, because Asp101 is modified followed by modifications of other carboxyls. In this case, we suggest that the amine (Melphalan) may also bind to the substrate binding site in competition with EDC. With this type of amine, enzyme-nucleophile interactions predominate, and the selective activation of Asp101 by EDC is reduced to lead a more random reaction.