Biomedical subjects

F Berends

Publications and source records attributed to F Berends.

At least 19 recordsLinked to original sources

PubMed2005

The age of stored red blood cell concentrates at the time of transfusion.

In the past decade, studies suggesting a reduced oxygen delivery by stored red blood cell concentrates (RBCCs) have initiated a discussion about the use of fresh versus old blood. We determined whether old RBCCs represent a significant part of the total of RBCCs issued. The age of RBCCs at the time of transfusion was determined in 74 084 units during a 5-year period in the Academic Medical Center, a main Dutch University Hospital. The mean (+/-SD) storage time of the total number of transfused RBCC was 19.4 +/- 7.0 days, and 37% were older than 3 weeks. As more than one-third of the transfused RBCC units are stored for longer than 3 weeks, the research examining differences in oxygen delivery between fresh and stored RBCC is relevant for packed RBC transfusion practice.

Blood Preservation↗

PubMed2001

Decreased circulating levels of von Willebrand factor after intravenous administration of a rapidly degradable hydroxyethyl starch (HES 200/0.5/6) in healthy human subjects.

OBJECTIVE: Impairment of haemostasis has been described with slowly degradable medium molecular weight hydroxyethyl starch (MMW-HES), whereas rapidly degradable MMW-HES is generally considered to have no important effects on blood coagulation. This study was undertaken to investigate the effects of a rapidly degradable MMW-HES plasma substitute on primary haemostasis and blood coagulation in human subjects. DESIGN: Randomised, cross-over study. SETTING: Research unit of a university hospital. PARTICIPANTS: Nine healthy, adult male volunteers. INTERVENTIONS: A 60-min intravenous infusion of 1 l HES 200/0.5/6 (HAES-steril 6%) or 4% albumin (control). MEASUREMENT AND RESULTS: The infusion of HES resulted in decreased circulating levels of von Willebrand factor antigen (from 85+/-8% to 59+/-6% after HES vs from 80+/-7% to 69+/-8% after albumin, p<0.05) and ristocetin cofactor activity (from 93+/-4 to 67+/-4% after HES vs from 79+/-5 to 75+/-5% after albumin, p<0.01). This was associated with an impairment of in vitro platelet function as determined with the PFA-100 platelet function analyser (closure time with collagen/epinephrine from 120+/-7 to 159+/-14 s after HES vs from 121+/-7 to 137+/-10 s after albumin, p<0.05; with collagen/ADP from 88+/-3 to 116+/-9 s and from 103+/-4 to 114+/-7 s after HES and albumin, respectively, p=0.01). CONCLUSIONS: The infusion of 1 l of HES 200/0.5/6 in healthy human subjects results in moderately decreased plasma levels of von Willebrand factor associated with impairment of platelet function.

Adult↗

PubMed2000

Laparoscopic elective treatment of diverticular disease. A comparison between laparoscopic-assisted and resection-facilitated techniques.

BACKGROUND: Because of the presence of significant inflammatory reaction, elective surgical laparoscopic-assisted treatment of complicated diverticular disease can be difficult, leading to a high conversion and complication rate. Laparoscopic alternatives to this assisted approach consist of the hand-assisted method and the more conventional facilitated laparoscopic sigmoid resection. Facilitated laparoscopic sigmoid resection implies laparoscopic mobilization of the sigmoid as much as possible and splenic flexure when called for. Through a Pfannenstiel incision, the difficult steps of the operation-such as the dissection of the inflammatory process and taking down the fistula, but also resection and manual anastomosis-can be performed. In this study, we compare the operating time, conversion rate, complications, and costs of both assisted and resection-facilitated techniques. METHODS: We compared two consecutive series of 35 patients with diverticular disease who underwent a sigmoid resection by laparoscopy. Both groups were comparable in terms of age, gender, and kind of complicated diverticular disease. RESULTS: The operating time, conversion rate, and costs were all less in the laparoscopic-facilitated group. The fact that there were no conversions in this group is the most important finding of this study. Not only was it possible to convert from the assisted laparoscopic approach to laparotomy (five patients of 35), it was also possible to convert from the assisted to the facilitated form (seven of 35 patients). CONCLUSIONS: Laparoscopic-facilitated sigmoid resection is a feasible intervention for all forms of complicated diverticular disease and yields marked reductions in operating time, conversion rate, and operative and general costs.

Aged↗

PubMed2000

Monitoring therapy with vitamin K antagonists in patients with lupus anticoagulant: effect on different tests for INR determination.

BACKGROUND: Lupus anticoagulant (antiphospholipid antibodies) is associated with venous and arterial thrombosis in patients with and without autoimmune disorders. Vitamin K antagonists are the treatment of choice in patients with thrombosis, of which the dose is titrated by INR monitoring. Several recent reports suggest that the presence of the lupus anticoagulant disturbs the INR test and may lead to unreliable results with a large variation in INR values, dependent on the reagents used. METHODS: We studied 11 lupus anticoagulant positive patients and 11 lupus anticoagulant negative patients, all using vitamin K antagonists. The INR value was determined using seven different tests and the variation in INR values was compared between the two groups. The amidolytic Factor X levels were used as an phospholipid independent measure for intensity of warfarin therapy. Factor VII and X activity were measured to assess the stability of warfarin therapy. RESULTS: The variation of the results with different INR tests within one patient was minimal and comparable in the two groups for INR's in the therapeutic range. The coefficient of variation for the cases and control group was 10.43 and 9.35, respectively. Variation in both groups increased at supratherapeutic levels of anticoagulation and when the anticoagulation was unstable (measured with Factor X/Factor VII ratio). The relationship between INR values and Factor X analysis revealed no influence of the lupus anticoagulant. CONCLUSIONS: In this study, lupus anticoagulant antibodies do not disturb INR laboratory tests. Differences in INR measurements are seen in patients with a high intensity of anticoagulation and in patients who either just started or in whom no stable anticoagulation has been achieved. Abbreviated Abstract. This study investigates the influence of lupus anticoagulant on INR determination tests in patients treated with warfarin. Eleven cases and eleven lupus anticoagulant negative control patients, also on warfarin therapy, were included. Seven INR results per patient were obtained using different laboratory tests. A factor X assay was performed to obtain an independent measure for the intensity of warfarin therapy. The variation of INR results between the cases and controls revealed no difference in these groups. In addition, the relationship between INR values and Factor X analysis indicated no influence of the lupus anticoagulant. What was observed was an increased difference in INR values in patients with a high intensity of anticoagulation and in patients who either just started or in whom no stable anticoagulation has been achieved

Factor VII↗

PubMed1998

The formation and repair of cisplatin-DNA adducts in wild-type and cisplatin-resistant L1210 cells: comparison of immunocytochemical determination with detection in isolated DNA.

We have studied the formation and repair of cisplatin-DNA adducts in wild-type mouse leukemia L1210/0 cells and in the sublines L1210/2 and L1210/5, which differ in cisplatin sensitivity. In a colony-formation assay these sublines were 9- and 22-fold more resistant compared to L1210/0, respectively. Cisplatin-induced DNA modification was studied at the cellular level by immunocytochemistry with antiserum NKI-A59 raised against cisplatin-treated DNA. Levels of nuclear staining immediately after a 1-h treatment were similar to those seen after a 24-h post-incubation in drug-free medium. Clear differences in DNA platination were found between the cell lines: immediately after exposure, L1210/2 and L1210/5 showed only 32 and 14%, respectively, of the nuclear staining observed in L1210/0, and 48 and 13% after 24 h. In these experiments, adduct-specific nuclear staining was quantified as the area under the adduct versus concentration curves (AUC). The formation and repair in these cell lines of the bifunctional adducts cis-Pt(NH3)2d(pGpG) (Pt-GG), cis-Pt(NH3)2d(pApG) (Pt-AG) and cis-Pt(NH3)2(dGMP)2 (G-Pt-G) were studied with an enzyme-linked immunosorbent assay (ELISA). No relation between repair and resistance was observed. The results suggest that differences in induced DNA platination levels, rather than in repair, are responsible--at least in part--for the differences in cisplatin resistance. A mechanism such as an increased tolerance of the resistant cells to plantinum-DNA damage may also be involved.

Animals↗

PubMed1998

DNA adducts, mutant frequencies, and mutation spectra in various organs of lambda lacZ mice exposed to ethylating agents.

To investigate tissue-specific relations between DNA adducts and mutagenesis in vivo, lambda lacZ transgenic mice were treated i.p. with N-ethyl-N-nitrosourea (ENU), diethylnitrosamine (DEN), and ethyl methanesulphonate (EMS). In liver, bone marrow, and brain DNA from mice sacrificed at several time points after treatment O6-ethylguanine (O6-EtG) and N7-ethylguanine (N7-EtG) levels were determined as well as the mutant frequency (MF) in lacZ. In liver DNA of ENU- and DEN-treated mice, the bulk of O6-EtG was removed at 3 days after treatment, while the MF continued to increase thereafter. This suggests that O6-EtG is not the major premutagenic lesion in the liver. Indeed, sequence analysis of mutants showed only 24% GC-->AT transitions, consistent with the O6-EtG lesion, and 28% TA-->AT transversions, expected from O2-ethylthymine. In bone marrow after ENU treatment, a maximum mutation induction occurred at 3 days post-treatment, of which 43% were GC-->AT mutations and 22% were TA-->AT mutations. This suggests that in bone marrow O6-EtG may be a major premutagenic lesion at the 3-day time point. In liver and bone marrow, EMS treatment gave rise to a high level of N7-EtG and a low level of O6-EtG but no increase in MF. No adducts or mutation induction were observed in bone marrow of DEN-treated mice. No MF increase was observed in the brain of either ENU- or EMS-treated mice, although O6- and N7-adducts were present.

Animals↗

PubMed1998

Impaired haemostasis by intravenous administration of a gelatin-based plasma expander in human subjects.

The aim of this study was to investigate the effects of a gelatin-based plasma expander on blood coagulation and haemostasis in human subjects. Six healthy men were studied in a randomised, controlled cross-over study to investigate the effects of a 60 min intravenous infusion of either 1 l gelatin-based plasma substitute (Gelofusine) or 0.9% NaCl (control). The infusion of gelatin resulted in a 1.7 fold increase in bleeding time at 60 min and a 1.4 fold increase at 120 min, while saline had no effect (p <0.05). Aggregation studies revealed a significant impairment of ristocetin-induced platelet aggregation (p <0.05), associated with a substantial decrease of vWF:ag (-32% vs. -5%, p <0.05) and ristocetin co-factor (-29% vs. +1%, p <0.05) and without in vitro impairment of the platelet glycoprotein 1b receptor. Gelatin caused a decrease in thrombin-antithrombin complexes (-45% vs. -4%, p <0.05) and F1+2 (-40% vs. +1%, p <0.05). The decrease in circulating levels of vWF:ag, vWF R:Co, thrombin-antithrombin complexes and F1+2 was more than could be expected by the calculated plasma-dilution generated by Gelofusine. Our results demonstrated that the administration of a gelatin-based plasma substitute results in a significant impairment of primary haemostasis and thrombin generation. The defect in primary haemostasis appears to be related to a gelatin-induced reduction in von Willebrand factor, whereas the decreased thrombin generation may be due to the dilution of coagulation factors induced by Gelofusine.

Adult↗

PubMed1997

Coagulation abnormalities in type 1 Gaucher disease are due to low-grade activation and can be partly restored by enzyme supplementation therapy.

In type 1 Gaucher disease a bleeding tendency occurs which is partly caused by thrombocytopenia due to massive splenomegaly. In addition, low levels of factors IX and XI have been described. The mechanism responsible for these clotting factor abnormalities is unknown. We performed a detailed study of parameters of coagulation and fibrinolysis in 30 type 1 Gaucher disease patients (14 splenectomized) before and after treatment with enzyme supplementation therapy. Pre-treatment aPTT and PT were prolonged in 42% and 38% of patients, respectively. In 30-60% serious deficiencies (< 50%) of coagulation factors XI, XII, VII, X, V and II were observed. The low levels of factor V correlated with platelet count and were inversely associated with splenic volume. Levels of inhibitors were mildly decreased. Markers for activation of coagulation (thrombin-antithrombin (TAT) complex) and fibrinolysis (PAP complex, fibrin cleavage product D-dimer) were significantly elevated, especially in the splenectomized patients, indicating ongoing activation of these processes. After 12 months of enzyme supplementation therapy partial correction occurred. Thus, severe disorders of the coagulation system occur in Gaucher disease, contributing to the bleeding tendency. The deficiencies may be the result of consumption of coagulation factors caused by ongoing low-level coagulation activation. possibly due to mononuclear cell activation.

Adult↗

PubMed1996

Formation and persistence of O6-ethylguanine in genomic and transgene DNA in liver and brain of lambda(lacZ) transgenic mice treated with N-ethyl-N-nitrosourea.

LacZ transgenic mice are suitable for short-term mutagenicity studies in vivo. Mutagenicity in these mice is determined in the lacZ transgene. Since the lacZ gene is of bacterial origin the question has been raised whether DNA-adduct formation and repair in the transgene are comparable to those in total genomic DNA. Mice were treated with N-ethyl-N-nitrosourea (ENU) and killed at several time points following treatment. Some mice were pretreated with O6-benzylguanine to inactivate the repair protein O6-alkylguanine-DNA alkyltransferase (AGT). O6-ethylguanine (O6-EtG) was determined in lacZ in liver and brain by means of a monoclonal antibody-based immunoaffinity assay. In addition, O6-EtG and N7-ethylguanine (N7-EtG) were assayed in total genomic DNA of liver and brain with an immunoslotblot procedure. In liver, the initial O6-EtG level in total genomic DNA was 1.6 times that in lacZ. The extent of repair of O6-EtG during the first 1.5 h after treatment was 2.1 times that in lacZ. At later time points, O6-EtG repair was the same. N7-EtG repair in genomic DNA was evident. In contrast to the liver, little repair of O6-EtG in total genomic and lacZ DNA occurred in the brain while N7-EtG was repaired. No initial difference in O6-EtG levels were found in lacZ and genomic brain DNA. These findings indicate that in the liver, total genomic DNA is more accessible than lacZ to ENU and/or the AGT protein, during the first 1.5 h following treatment. Because the difference in O6-EtG levels in the transgene and genomic DNA in the liver is restricted to the first 1.5 h after treatment, while the fixation of mutations occurs at later time points, O6-EtG-induced mutagenesis most likely is also very similar in both types of DNA.

Animals↗

PubMed1995

Formation of DNA adducts by the anticancer drug carboplatin: different nucleotide sequence preferences in vitro and in cells.

We have studied the formation of adducts upon carboplatin treatment of isolated DNA and in cells. The major adduct formed in vitro, determined with atomic absorption spectroscopy and enzyme-linked immunosorbent assay, was the intrastrand cross-link cis-Pt(NH3)2d(pGpG)(Pt-GG) (58%). cis-Pt-(NH3)2d(pApG) (Pt-AG) (11%), cis-Pt(NH3)2d(GMP)2 (G-Pt-G) (9%), and monofunctionally bound platinum (cis-Pt(NH3)3dGMP (Pt-G), 22%) were formed in smaller amounts. These relative occurrences of the adducts, average values found between 1 and 16 h of incubation, are comparable with those after incubation with cisplatin. The formation of carboplatin-DNA adducts was slow, and about 230-fold more carboplatin than cisplatin (molar dose) was required to obtain equal levels of platination after 4 h of incubation. However, less than 20 times more carboplatin was needed to obtain equal levels of cytotoxicity after 1 h of exposure of CHO cells. The percentages of the carboplatin-DNA adducts after 7-12 h postincubation of the cells (determined with ELISA), Pt-GG (30%), Pt-AG (16%), G-Pt-G (40%), and Pt-G (14%), were different from those of the in vitro data. After 12 h postincubation, the number of interstrand cross-links (determined by alkaline elution) amounted to about 10% of the G-Pt-G adducts and 3-4% of the total amount of adducts. The immunocytochemical detection (with antiserum NKI-A59) of the platinum-DNA modifications showed a pattern similar to that found for the various bifunctional adducts: the initially low levels slowly increased to maximum values within 7-12 h and then slowly decreased. In conclusion, carboplatin forms the same bifunctional adducts as cisplatin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Effects of thiourea and ammonium bicarbonate on the formation and stability of bifunctional cisplatin-DNA adducts: consequences for the accurate quantification of adducts in (cellular) DNA.

Cisplatin reacts with DNA by forming mainly bifunctional adducts via reactive monofunctional intermediates. When freshly platinated DNA was postincubated with thiourea (10 mM, at 23 or 37 degrees C) for periods of up to 24 h, followed by determination of mono- and diadducts, a rapid initial decrease was seen in the fraction of diadducts, followed by a much slower decrease. About 40% diadducts were found after 10-min postincubation at 23 degrees C, which dropped to some 14% after 24 h at 37 degrees C; total platination was hardly affected. Postincubation of "aged" platinated DNA (no reactive monoadducts) only showed the slower decrease. The rapid process is likely to represent monoadduct inactivation, preventing the formation of diadducts, whereas the slow reaction must be interpreted as diadduct-to-monoadduct conversion. Similar reactions, but less efficient than with thiourea, occurred during dialysis against NH4HCO3 (0.1-1 M). Pt-containing (di)nucleotides in digested DNA were hardly affected by thiourea. Rapid reduction of the measured level of bifunctional adducts also occurred when cisplatin-treated Chinese hamster ovary cells were postincubated with thiourea, with concomitant increase in survival. It is concluded that quantification of the real levels of mono- and diadducts in freshly platinated DNA requires a posttreatment with thiourea of 30-60 min at 37 degrees C.

Animals↗

PubMed1995

Cisplatin- and carboplatin-DNA adducts: is PT-AG the cytotoxic lesion?

In order to determine the nature of the cytotoxic lesion(s) formed by the antitumour drugs cisplatin and carboplatin, a comparative study was made of bifunctional DNA-adduct formation by these drugs. The kinetics of bifunctional cisplatin adduct formation were studied with DNA in vitro and in cultured Chinese hamster ovary (CHO) cells. Prior to adduct measurements with AAS in in vitro platinated DNA and with ELISA in cellular DNA, the monoadducts were inactivated with thiourea (10 mM; 1 h at 37 degrees C). The data indicated that the conversion of monofunctional to bifunctional adducts, with t1/2 of approximately 2 h (37 degrees C), leads to maximum intrastrand adduct levels after approximately 4-6 h postincubation. This interval coincided with the period during which the cytotoxic effect of cisplatin could be reduced by a 1 h 10 mM thiourea post-incubation of the cells. The formation of interstrand crosslinks continued for approximately 7 h of post-incubation; then these amounted to approximately 2% of the total DNA adducts. When a DNA sample was dialysed against 0.1 M NH4HCO3 (16 h, 37 degrees C) immediately after cisplatin treatment, in order to block mono- to bifunctional adduct conversion, adduct levels were found similar to those after the 4-6 h post-incubation. From this it is clear that the high values reported earlier for bifunctional cisplatin adducts in such DNA samples are not correct. These values apparently represent the amounts of adducts that eventually would have been formed during post-incubation in DNA in vitro but also in cells in the absence of cellular repair. The cisplatin data of CHO cells were compared with those after treatment of the cells with equitoxic doses of carboplatin. The data indicate that after 12 h post-incubation, when all bifunctional adducts are formed, the total amount of the various bifunctional adducts after cisplatin treatment (37.5 +/- 4.5 fmol/micrograms DNA) was in the same range as that after carboplatin (32.8 +/- 6.3 fmol/micrograms DNA). However, because the relative occurrences of the adducts were different, it could also be concluded that if one of the diadducts were exclusively responsible for the cytotoxic effect of these platinum antitumour drugs, Pt-AG is the only likely candidate.

Animals↗

PubMed1994

Association of idiopathic venous thromboembolism with single point-mutation at Arg506 of factor V.

Abnormal coagulation factor V may underlie the thrombotic events associated with resistance to activated protein C (APC). We analysed 27 consecutive patients with documented idiopathic (recurrent) thromboembolism for the occurrence of point mutations within the APC sensitive regions of blood coagulation factor V. In 10 patients we observed a single basepair mutation resulting in a substitution of Arg506 to Gln. This mutation was significantly linked to in-vitro resistance to APC in these subjects. This mutation at Arg506 of factor V may form the molecular basis for the thrombotic events associated with APC resistance.

Adult↗

PubMed1994

Monitoring anticoagulant therapy by activated partial thromboplastin time: hirudin assessment. An evaluation of native blood and plasma assays.

BACKGROUND: Recombinant hirudin (RH) is a new anticoagulant for prophylaxis and treatment of venous and arterial thrombosis. To which extent the activated partial thromboplastin time (APTT) is suitable for monitoring of RH has not been properly evaluated. Recently, a capillary whole blood device was developed for bed-side monitoring of the APTT and it was demonstrated that this device was suitable to monitor heparin therapy. However, monitoring of RH was not evaluated. STUDY OBJECTIVES: To evaluate in vitro and ex vivo the responsiveness and reproducibility for hirudin monitoring of the whole blood monitor and of plasma APTT assays, which were performed with several reagents and two conventional coagulometers. RESULTS: Large interindividual differences in hirudin responsiveness were noted in both the in vitro and the ex vivo experiments. The relationship between the APTT, expressed as clotting time or ratio of initial and prolonged APTT, and the hirudin concentration was nonlinear. A 1.5-fold increase of the clotting times was obtained at 150-200 ng/ml plasma. However, only a 2-fold increase was obtained at hirudin levels varying from 300 ng to more than 750 ng RH/ml plasma regardless of the assays. The relationship linearized upon logarithmic conversion of the ratio and the hirudin concentration. Disregarding the interindividual differences, and presuming full linearity of the relationship, all combinations were equally responsive to hirudin. CONCLUSIONS: All assays were equally responsive to hirudin. Levels up to 300 ng/ml plasma can be reliably estimated with each assay. The manual device may be preferable in situations where rapid availability of test results is necessary.

Adult↗

PubMed1991

Induction and disappearance of DNA strand breaks and/or alkali-labile sites in human lymphocytes exposed to N-ethyl-N-nitrosourea.

We investigated the induction and disappearance of DNA lesions that are detected as single-strand breaks (SSBs) with the alkaline filter elution technique, in human peripheral blood lymphocytes (PBLs) following exposure to N-ethyl-N-nitrosourea (ENU). In PBLs of the 15 individuals studied, 35 +/- 16% (mean +/- SD) of the SSBs present at the end of a 20 min treatment disappeared within 1 h; up to 24 h post-treatment no further disappearance was observed. Interindividual differences in SSB disappearance were considerable; in two cases, almost no SSBs disappeared over the 1 h period. In PBLs of the same 15 individuals 85 +/- 3% (mean +/- SD) of the SSBs induced by 4 Gy of gamma-rays were found to disappear within 1 h, which indicated no interindividual differences. Multiple blood sampling at 1 month intervals indicated that interindividual differences in ENU-induced SSB repair are constant and do not vary with time. The low rate of SSB disappearance appeared to correlate with low cell survival after ENU exposure, measured as a reduction in phytohemagglutinin-stimulated incorporation of [3H]-thymidine. The cytosine analog 1-beta-D-arabinofuranosyl-cytosine completely inhibited SSB disappearance, indicating that the removal of the repairable ENU-induced SSBs involves excision repair events.

Cell Survival↗

PubMed1990

Kinetics of the formation and removal of cisplatin-DNA adducts in blood cells and tumor tissue of cancer patients receiving chemotherapy: comparison with in vitro adduct formation.

During chemotherapy with a cisplatin-containing combination of drugs, 217 blood samples from 30 cancer patients were analyzed for the presence of the main cisplatin-DNA adduct cis-Pt(NH3)2d(pGpG) (Pt-GG). Cisplatin was administered during 3-h infusions on each of 5 consecutive days, resulting in increasing adduct levels which, on the average, were about twice as high after the fifth as after the first infusion. Higher levels were found in blood samples of patients who received the same total amount of cisplatin in one single 3-h infusion. No significant differences in adduct levels were found during first and repeated courses. The nonlinear dependence of adduct levels on total dose can be attributed to removal of adducts. At 21 h after a very first cisplatin infusion 76% of the adducts were removed. Lower percentages of removal were observed over the 21-h periods following the fourth and fifth infusions of 5-day courses (49 and 53%, respectively). After the initial 21 h the removal of adducts continued, albeit at a slower rate. Substantial interindividual variation was found in the adduct levels, which did correlate with the levels obtained after in vitro cisplatin treatment of blood samples from the same patients but not with their age or gender. Testicular cancer patients with complete tumor response showed higher adduct levels in their blood than those with partial response or progressive disease. When blood samples from 8 healthy volunteers were treated with cisplatin in vitro, the person-to-person variation in adduct levels and the intraindividual variation observed over a 2-year period were found to be in the same range, which was narrower than that observed with samples from treated patients. In vitro studies with human blood showed that the formation of the Pt-GG adduct is proportional to cisplatin concentration and complete after about 1 hour. In some of the in vivo and in vitro cisplatin-treated blood samples, all 4 known platinum-DNA adducts were determined. In all cases Pt-GG was by far the major adduct, and no significant differences were observed with respect to the relative amounts of the 4 adducts. Similar adduct ratios were found in DNA from a testicular tumor obtained from a patient who underwent orchidectomy; the Pt-GG adduct level was about 10-fold higher than that in his blood cells.

Adult↗

PubMed1989

Efficient rescue of integrated shuttle vectors from transgenic mice: a model for studying mutations in vivo.

To study gene mutations in different organs and tissues of an experimental animal, we produced transgenic mice harboring bacteriophage lambda shuttle vectors integrated in the genome in a head-to-tail arrangement. As a target for mutagenesis, the selectable bacterial lacZ gene was cloned in the vector. The integrated vectors were rescued from total genomic DNA with high efficiency by in vitro packaging and propagation of the phages in a LacZ- strain of Escherichia coli C. The background mutation frequencies in brain and liver DNA appeared to be low, as was indicated by the absence of colorless plaques among 138,816 and 168,160 phage isolated from brain and liver DNA, respectively. Treatment of adult female transgenic mice with N-ethyl-N-nitrosourea resulted in a dose-dependent increase of the frequency of mutated vectors isolated from brain DNA, up to 7.4 x 10(-5) at 250 mg of the alkylating agent per kilogram of body weight. At this dose, in liver DNA of the same mice, mutation frequencies were approximately 3 x 10(-5). DNA sequence analysis of four mutant vectors isolated from brain DNA indicated predominantly G.C----A.T transitions. These results demonstrate the value of this transgenic mouse model in studying gene mutations in vivo. In addition to its use in fundamental research, the system could be used as a sensitive, organ-specific, short-term mutagenicity assay.

Animals↗

PubMed1989

Influence of the degree of DNA modification on the immunochemical determination of cisplatin-DNA adduct levels.

Two different enzyme-linked immunosorbent assays (ELISAs) are used for the sensitive determination of cisplatin-DNA adducts in, for example, blood cells of cisplatin-treated cancer patients. Poirier et al. determined the adducts in native DNA with an antiserum raised against highly modified cisplatin-DNA. Fichtinger-Schepman et al. assayed the various adducts after chromatography of enzymatically digested DNA samples, with antibodies raised against synthetic haptens mimicking the Pt-containing digestion products. In identical human samples analysed by both methods, 14- to 300-fold higher adduct levels were found with the Fichtinger-Schepman method. Adduct levels in organ samples of cisplatin-treated rats and mice allowed comparison of both ELISAs with independent Pt assays (atomic absorption spectroscopy, AAS). AAS data confirmed the results of the Fichtinger-Schepman assay, whereas ELISAs according to Poirier showed differences up to a factor of 1000. Analysis of cisplatin adducts in in vitro modified DNA revealed that the Poirier antibodies yield correct estimates with highly modified DNA, but fall short at low platination levels: no adducts could be detected in samples modified to a level similar to that found in blood cells from cisplatin-treated patients. The positive responses obtained with samples of such patients, who had been treated repeatedly, might be explained if cisplatin adducts are preferentially formed around persistent lesions from the previous treatments.

Animals↗