Toxicological results in a fatal and two non-fatal cases of scopolamine-facilitated robberies.

The use of scopolamine as an incapacitating drug, in sexual crimes and robberies, has been known for many decades. However, blood concentrations and doses of scopolamine in those cases are largely unknown. Here we present the toxicological results of one fatal and two non-fatal cases in a series of scopolamine-facilitated robberies. In the fatal case, the concentration of scopolamine in heart blood was 0.30mg/L, about 3000 times higher than the average therapeutic level of 0.0001mg/L (for one dermal patch). In femoral blood, the concentration of scopolamine was much lower (0.0048mg/L), but still 50 times higher than therapeutic levels. The scopolamine concentration in the stomach was very high (20mg/kg) as compared to the heart blood and femoral blood, which explains the very high concentration in heart blood by postmortem leakage from the stomach. In the non-fatal case, the scopolamine concentration in serum, obtained 23h after the incident, was 0.00035mg/L. The estimated concentration of scopolamine at the time of the incident is 0.0035mg/L. In the other non-fatal case, scopolamine was detected in urine and in hair.

The relation between the blood benzodiazepine concentration and performance in suspected impaired drivers.

Several experimental studies have shown a negative influence of benzodiazepines on driving skills. The objective of this study is to study the relationship between the blood concentration of benzodiazepines and the influence on performance in field sobriety tests. A retrospective case file evaluation was conducted to select cases of drivers, tested positive for benzodiazepines only in the period from January 1999 to December 2004. Drivers were grouped into the categories sub therapeutic, therapeutic or elevated concentrations. The outcome of the tests (walking, walking after turn, nystagmus, Romberg's test, behavior, pupils and orientation) was binomial. A Chi square test was used to assess differences in proportions of the categorized cases. In total 171 cases were included. Observations of behavior (n=137; p<0.01), walking (n=109; p<0.01), walking after turn (n=89; p=0.02) and Romberg's test (n=88; p<0.05) were significantly related to the benzodiazepine concentration. There was no significant relation between benzodiazepine concentration and effect on pupil size, nystagmus or orientation. The results of our study indicate a relation between the concentration of benzodiazepines and the results of some performance tests. More effort is needed to standardize the tests and to determine the sensitivity and selectivity of the tests for benzodiazepines.

The relation between the use of psychoactive substances and the severity of the injury in a group of crash-involved drivers admitted to a regional trauma center.

OBJECTIVE: There is much evidence that driving under the influence of alcohol and/or drugs of abuse is related to an increased accident risk. A remaining question is whether the use of psychoactive substances is also related to clinically more severe accidents. The aim of this study is to explore the relationship between the use of psychoactive substances and the injury severity in a group of crash-involved drivers. METHODS: The study group included all injured car drivers, admitted to the regional trauma center, in the period from May 2000 until August 2001. The outcome of interest was the severity of injury, measured by using the Injury Severity Score (ISS). The determinant was the presence of psychoactive substances in blood and urine samples. Psychoactive substances tested for were alcohol, amphetamines, barbiturates, benzodiazepines, cannabis, methadone, opiates, and tricyclic antidepressants in blood and urine. RESULTS: The number of injured car drivers included in this study was 106. Overall, 43% (46/106) of the drivers tested positive for at least one psychoactive substance. Comparison of the means of the log ISS suggests that there is no significant difference between drivers who tested positive for alcohol and/or drugs, compared to drivers tested negative. CONCLUSION: The results of this study support the hypothesis that there is no clear association between use of psychoactive substances and the severity of crash-related injury.

Comparison of urine and oral fluid as matrices for screening of thirty-three benzodiazepines and benzodiazepine-like substances using immunoassay and LC-MS(-MS).

Benzodiazepines are the most frequently detected medicinal drugs in drivers. The use of benzodiazepines is associated with an increased road accident risk. In this study, the presence of benzodiazepines detected by liquid chromatography-(tandem) mass spectrometry [LC-MS(-MS)] in oral fluid and urine samples obtained from drivers stopped during a roadside survey was compared. In addition, the sensitivity and selectivity of enzyme multiplied immunoassay technique (EMIT II Plus) relative to LC-MS(-MS) was determined for both matrices. A total number of 1,011 urine samples were collected and screened for benzodiazepines using immunoassay (IA) (EMIT II Plus; cutoff 300 ng/mL). In the IA-positive (n = 25) and a group of randomly selected negative urine samples (n = 79), the presence or absence of benzodiazepines was confirmed by LC-MS-MS after deglucuronidation. The corresponding oral fluid samples (n = 101, 3 samples omitted), were analyzed by LC-MS(-MS) and IA (EMIT II Plus; cutoff 10 ng/mL). The presence of benzodiazepines was demonstrated by LC-MS-(MS) in all IA-positive urine samples, but in only four corresponding oral fluid samples. Concentrations in oral fluid were, one substance excepted, lower than in urine. The sensitivity and specificity of EMIT II Plus were better by using urine as matrix for screening of benzodiazepines than by using oral fluid. The results show that benzodiazepines are detectable in oral fluid. More research has to be done to determine the pharmacokinetic profile of the different benzodiazepines in oral fluid and to study the relationship between dose, concentration (in oral fluid and blood), and impairment.

Drug use and the severity of a traffic accident.

Several studies have showed that driving under the influence of alcohol and/or certain illicit or medicinal drugs increases the risk of a (severe) crash. Data with respect to the question whether this also leads to a more severe accident are sparse. This study examines the relationship between the use of alcohol, illicit drugs and/or medicinal drugs and the severity of an accident within a group of drivers that were involved in a crash in The Netherlands. Blood samples of 993 drivers, collected in the period from October 1998 through September 1999, were linked to accident characteristics as available from the National Transport Research Centre. The outcome measure was the severity of the accident. An accident was considered severe when the accident had resulted in hospital admission or death. All the blood samples obtained after the accident were screened for the presence of alcohol, illicit drugs (opiates, amphetamines and amphetamine-like substances, cocaine and metabolites, methadone, cannabinoids) and medicinal drugs (benzodiazepines, barbiturates and tricyclic antidepressants). The strength of the associations between exposure to the different classes of alcohol/drugs/medicines and the severity of the accident was evaluated using logistic regression analysis and were expressed as odds ratios (OR), adjusted for age, gender, time of the day, day of the week and urban area. The most frequently detected drugs were cannabinoids, benzodiazepines and cocaine. Our results showed no clear association between the use of alcohol, illicit drug and/or medicinal drug use and the severity of the accident. Given the process of obtaining blood samples from drivers involved in accidents and the retrospective nature of the study, we cannot rule out the occurrence of selection bias. Therefore, our findings need further confirmation.

Quantitative analysis of 33 benzodiazepines, metabolites and benzodiazepine-like substances in whole blood by liquid chromatography-(tandem) mass spectrometry.

A quantitative method using high-performance liquid chromatography-mass spectrometry (LC-MS, ion trap) after matrix supported liquid-liquid extraction is described for the simultaneous determination in whole blood of 33 benzodiazepines including metabolites and benzodiazepine-like substances. The limits of detection (LOD) range from 0.0001 to 0.0126 mg/l. Linearity is satisfactory for all compounds. The extraction recoveries for the benzodiazepines in whole blood are between 60 and 91%, desmethyldiazepam, OH-bromazepam and brotizolam excepted. Selectivity, accuracy and precision are satisfactory for clinical and forensic purposes.

The detection and identification of quaternary nitrogen muscle relaxants in biological fluids and tissues by ion-trap LC-ESI-MS.

Quaternary nitrogen muscle relaxants pancuronium, rocuronium, vecuronium, gallamine, suxamethonium, mivacurium, and atracurium and its metabolites were extracted from whole blood and other biological fluids and tissues by using a solid-phase extraction procedure. The extracts were examined by using high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). The drugs were separated on a ODS column in a gradient of ammonium acetate buffer (pH 5.0) and acetonitrile. Full-scan mass spectra of the compounds showed molecular ions, and MS-MS spectra showed fragments typical of the particular compounds. LC-ESI-MS allowed an unequivocal differentiation of all muscle relaxants involved. The method was applied in a case of rocuronium and suxamethonium administration in a Caesarian section and in a case of intoxication by pancuronium injection. In both cases, the administered drugs could be detected and identified in the supplied samples.

Driving under the influence of alcohol and/or drugs in the Netherlands 1995-1998 in view of the German and Belgian legislation.

This study presents the test results of blood and urine samples of impaired drivers in the Netherlands between January 1995 and December 1998. In this period, the blood alcohol concentrations of 11,458 samples have been determined and 1665 blood or urine samples have been analysed for drugs. The median alcohol concentration was between 1.7 and 1.8 mg/ml blood. In 80% of the 1665 analysed samples drugs were detected. At least 42% (702/1665) of the impaired drivers were poly-drug users, with cocaine present in the most frequent combinations. In the Netherlands, the procedure to prove driving under the influence is complex. This procedure can be made more efficient and more effective by embedding the analytical test results, needed to prosecute an impaired driver, in the law. In Belgium and Germany, such laws already are in force. If we would apply the qualifications of the new Belgian law on our analytical data, 67% of the impaired drivers included in this comparison could have been prosecuted without discussion in court.

Use of clonidine for chemical submission.

CASE REPORT: An East-European prostitute in Amsterdam robbed several victims, after having sedated them with clonidine solution (available as plastic ampoules of eyedrops) added to her victims' drinks. One victim was hospitalized. His symptoms included bradycardia, hypotension, hypothermia, pallor, cyanosis, and impaired consciousness. Treatment included isoprenaline for 28 hours. The victim was released from hospital the next day. In court, the female offender confessed and was sentenced to prison for 3 1/2 years. She may have administered doses as high as 8 mg clonidine.

Redox cycling of potential antitumor aziridinyl quinones.

The formation of reactive oxygen intermediates (ROI) during redox cycling of newly synthesized potential antitumor 2,5-bis (1-aziridinyl)-1,4-benzoquinone (BABQ) derivatives has been studied by assaying the production of ROI (superoxide, hydroxyl radical, and hydrogen peroxide) by xanthine oxidase in the presence of BABQ derivatives. At low concentrations (< 10 microM) some BABQ derivatives turned out to inhibit the production of superoxide and hydroxyl radicals by xanthine oxidase, while the effect on the xanthine-oxidase-induced production of hydrogen peroxide was much less pronounced. Induction of DNA strand breaks by reactive oxygen species generated by xanthine oxidase was also inhibited by BABQ derivatives. The DNA damage was comparable to the amount of hydroxyl radicals produced. The inhibiting effect on hydroxyl radical production can be explained as a consequence of the lowered level of superoxide, which disrupts the Haber-Weiss reaction sequence. The inhibitory effect of BABQ derivatives on superoxide formation correlated with their one-electron reduction potentials: BABQ derivatives with a high reduction potential scavenge superoxide anion radicals produced by xanthine oxidase, leading to reduced BABQ species and production of hydrogen peroxide from reoxidation of reduced BABQ. This study, using a unique series of BABQ derivatives with an extended range of reduction potentials, demonstrates that the formation of superoxide and hydroxyl radicals by bioreductively activated antitumor quinones can in principle be uncoupled from alkylating activity.

Interactions between potential anti-tumour 2,5-bis(1-aziridinyl)-1,4-benzoquinone derivatives and glutathione: reductive activation, conjugation and DNA damage.

The interaction between glutathione and potential anti-tumour 3,6-disubstituted 2,5-bis(1-aziridinyl)-1,4-benzoquinone (BABQ) derivatives has been studied using u.v. spectrophotometry and h.p.l.c. The formation of BABQ-glutathione adducts was demonstrated in vitro for the BABQ parent compound (TW13), triaziquone (2,3,5-tris(1-aziridinyl)-1,4-benzoquinone) and for BABQ derivatives containing halogen substituents. The clinically-used BABQ derivative diaziquone (AZQ; 2,5-bis(1-aziridinyl)-3,6-bis(ethoxycarbonylamino)-1,4-benzoquinon e) did not react with glutathione. TW13 and triaziquone markedly inactivated bacteriophage M13-DNA in the presence of glutathione. This inactivation is probably produced by reductive activation of the BABQ derivative to a DNA-alkylating semiquinone radical. However, formation of bulky glutathione adducts decreases reactivity to DNA. Halogen-substituted BABQ derivatives react rapidly with glutathione to form adducts. This appeared to prevent DNA alkylation by these compounds. Comparison of these results with in vivo and in vitro activity against tumour models (L1210) suggests that in vivo halogen-substituted BABQ derivatives are efficiently inactivated by glutathione conjugation. The differences between the halogen-substituted BABQ derivatives on the one hand and TW13 and triaziquone on the other hand are probably caused by a difference in reaction mechanism with glutathione. From the viewpoint of drug design, halogen-substituted BABQ derivatives are expected to be inactive anti-tumour agents, in spite of high reactivity and activity in tumour models in vitro.

Reductive activation of potential antitumor bis(aziridinyl)benzoquinones by xanthine oxidase: competition between oxygen reduction and quinone reduction.

The reduction of a series of 2,5-bis(1-aziridinyl)-1,4-benzoquinone (BABQ) derivatives with various 3,6 substituents by the enzyme xanthine oxidase has been studied. The reduction rate has been assayed by measuring the rate of reduction of cytochrome c, which is very efficiently reduced by reduced BABQ species. Under nitrogen, the reduction rate correlated with the quinone reduction potential and steric parameters. Comparing reduction rates under nitrogen and air demonstrates that at BABQ concentrations greater than 25 microM the competition for electrons from xanthine oxidase between oxygen and the BABQ derivative is dominated by the latter. This is also confirmed by the effect of superoxide dismutase (SOD): in the presence of a BABQ derivative, cytochrome c reduction can be totally inhibited by SOD, although the required amount of SOD depends on the redox potential of the quinones. This indicates that SOD causes the equilibrium between semiquinone and superoxide to shift, resulting in a decrease of the semiquinone concentration. It is concluded that reduction by xanthine oxidase is a simple and effective method for reducing aziridinylbenzoquinones.

Covalent binding studies on the 14C-labeled antitumor compound 2,5-bis(1-aziridinyl)-1,4-benzoquinone. Involvement of semiquinone radical in binding to DNA, and binding to proteins and bacterial macromolecules in situ.

2,5-Bis(1-aziridinyl)-1,4-benzoquinone (BABQ) is a compound from which several antitumour drugs are derived, such as Trenimone, Carboquone and Diaziquone (AZQ). The mechanism of DNA binding of BABQ was studied using 14C-labeled BABQ and is in agreement with reduction of the quinone moiety and protonation of the aziridine ring, followed by ring opening and alkylation. The one-electron reduced (semiquinone) form of BABQ alkylates DNA more efficiently than two-electron reduced or non reduced BABQ. Covalent binding to polynucleotides did not unambiguously reveal preference for binding to specific DNA bases. Attempts to elucidate further the molecular structure of DNA adducts by isolation of modified nucleosides from enzymatic digests of reacted DNA failed because of instability of the DNA adducts. The mechanism of covalent binding to protein (bovine serum albumin, BSA) appeared to be completely different from that of covalent binding to DNA. Binding of BABQ to BSA was not enhanced by reduction of the compound and was pH dependent in a way that is opposite to that of DNA alkylation. Glutathione inhibits binding of BABQ to BSA and forms adducts with BABQ in a similar pH dependence as the protein binding. The aziridine group therefore does not seem to be involved in the alkylation of BSA. Incubation of intact E. coli cells, which endogenously reduce BABQ, resulted in binding to both DNA and RNA, but also appreciable protein binding was observed.

DNA alkylation and formation of DNA interstrand cross-links by potential antitumour 2,5-bis(1-aziridinyl)-1,4-benzoquinones.

A series of 3,6-substituted 2,5-bis(1-aziridinyl)-1,4-benzoquinone derivatives was shown to alkylate calf thymus DNA and to form DNA interstrand cross-links. Alkylation and cross-link formation were enhanced after electrochemical reduction of the compounds and increased with lower pH in the pH range from 4.5 to 8.0. Reduction especially shifts the pH at which cross-linking and alkylation occurs to higher values, which are more physiologically relevant. This shift is probably caused by the increase in pKa value of the aziridine ring after reduction of the quinone moiety. The inactivation of single-stranded bacteriophage M13mp19 DNA to form phages in an E. coli host, by the 3,6-unsubstituted parent compound 2,5-bis(1-aziridinyl)-1,4-benzoquinone (TW13) was dependent upon reduction and pH in a similar way as was alkylation. The compound in our series with the least bulky, 3,6-substituents, TW13, caused a high amount of cross-link formation. Compounds with methyl-substituted aziridine rings showed low cross-linking ability. Our results support the concept that the protonated reduced compound is the reactive species that alkylates DNA, and that steric factors play an important role in the reactivity towards DNA. A correlation is observed between the ability to induce DNA interstrand cross-links and inactivation of M13mp19 bacteriophage DNA. Cross-link formation was also demonstrated in E. coli K12 cells, where the compounds are reduced endogenously by bacterial reductases.

Potential anti-tumour 2,5-bis (1-aziridinyl)-1,4-benzoquinones: relationship between cytotoxicity against DNA-repair deficient E. coli, inactivation of bacteriophage M13mp19, and in vitro and in vivo anti-tumour activity.

A series of reductively activated 3,6 substituted 2,5-bis(aziridinyl)benzoquinones, designed as potential anti-tumour agents, was shown to interact with DNA. This was assayed by studying the colony forming ability of DNA-repair deficient E. coli K12 and the inactivation of bacteriophage M13mp19. The activities of the compounds as measured in the two assays show a good correlation. Reduction of the quinone compounds is essential for activity in the two assays. Reduction activates opening of the aziridine ring and formation of an alkylating species. The results suggest that steric factors are important in DNA adduct formation. Methylation of the aziridine rings decreases the activity. Bulky 3,6-substitutes also decrease the activity. No correlation is found between above-mentioned assays for DNA modification and in vivo activity towards tumour cells. It is possible that, as well as DNA, other cellular targets are involved. A correlation is observed between the DNA modifying activity and toxicity against L1210 cells in vitro. The relative high activity against L1210 cells of bromine substituted compounds is possibly related to alternative mechanisms in which bromine acts as an efficient leaving group.

Photosensitized irreversible binding of estrone to protein via a hydroperoxide intermediate: an explanation of (photo-) allergic side-effects of estrogens.

After irradiation (lambda greater than 425 nm) for 15 min of a solution of [4-14C]-estrone, albumin and the photosensitizer hematoporphyrin in phosphate buffer, more than 30% of the radioactivity could not be extracted. When the protein was added after irradiation, irreversible binding also occurred. Sephadex gel filtration showed that the radiolabel was bound to albumin as well as to the photosensitizer. A 10 beta-hydroperoxide is the reactive intermediate in this binding. Inasmuch as phenolic steroids coupled to proteins have been used for the induction of estrogenic-specific antibodies, the irreversible binding observed between estrone and albumin by photosensitization might be an explanation for (photo)allergic disorders associated with estrogens.