Rafał Skowronek

Prevalence of hydrogen cyanide and carboxyhaemoglobin in victims of smoke inhalation during enclosed-space fires: a combined toxicological risk

Background. Hydrogen cyanide (HCN) is one of the most toxic components of fire smoke, but insufficient attention is paid to its potential role as a cause of injury or death in victims (alive or dead) of enclosed-space fires. Objective. To analyse the prevalence of toxic HCN exposure in fire victims and factors that may influence its toxicity, particularly the co-presence of carbon monoxide (CO) and ethanol. Materials and methods. Blood samples from fire victims and persons rescued from fires were analysed. Results. A positive result for HCN (mean concentration 16.83 mg/l) was detected in blood samples from 169 of 285 fire-related deaths (59%). Ethanol was present in 91 (65%) of 139 samples with coincident presence of HCN and carboxyhaemoglobin (COHb). HCN (mean 4.0 mg/l) was also detected in 20 of 40 (50%) fire survivors. Discussion. The high prevalence of coincident CO and HCN in enclosed-space, fire-related deaths should alert clinicians to suspect toxic HCN exposure in all persons rescued from fire with signs and symptoms of respiratory distress. Conclusions. Medical procedures in persons rescued from enclosed-space fires, especially in the pre-hospital setting, should be augmented to cover the possibility of toxic HCN exposure, particularly in individuals who do not respond to standard supportive therapy. Likewise, post-mortem investigations should routinely include assays for HCN when determining probable cause of death.

Comments on 'Measuring insulin in human vitreous humour using LC-MS/MS' by Thevis et al.

We have read the article by Prof. Mario Thevis and his team titled ‘Measuring insulin in human vitreous humour using LC-MS/MS’ with great interest. Congratulations to the authors on the first successful mass-spectrometry-based analysis of post-mortem material related to an insulin poisoning case. It is another step forward in the post-mortem diagnostics of insulin overdose. The vitreous humour (also called a ‘vitreous body’) is a very valuable material for chemical-toxicological analyses and easy to obtain during autopsy. Its main advantage is anatomical isolation, useful especially in the case of advanced autolytic and putrefactive changes. Insulin is a very important peptide hormone in forensic toxicology for two reasons: (1) its casual or intentional overdose may be lethal and (2) it is also one of the most frequently used anabolic doping agents in sport. The case presented of a 55-year-old non-diabetic victim who died from an insulin overdose is quite interesting, especially because of the availability of ante-mortem blood samples. We have to remember that such a situation is very rare in routine forensic practice, however, where we may analyze only post-mortem biological material and non-biological specimens, like syringes, ampoules, vials, or remnants of the infusion solution and tubings, as in the commented article. The interpretation of insulin levels in the post-mortem biological material is very difficult. The number of published papers dealing with this problem is relatively low. The time of survival after insulin injection depends on many different factors: type of insulin (differentiated onset of action and insulin half-life), method of administration (injection or insulin infusion pump), localization of injection sites on the body (different rate of absorption), etc. The time of survival certainly influences the insulin levels detected in the post-mortem material. Unfortunately, in forensic practice, we do not usually know that time, because the cadavers are frequently found a long time after death, for example when the victim is living alone. The processes of anteand post-mortem elimination, distribution, and insulin transport across the blood-ocular barrier need to be studied more. Further to the Thevis et al.’ article, we want to shortly present our own, so far unpublished experiences in insulin determination in autopsy blood and vitreous humour. So far we have only presented this work during the 9th National Congress of the Polish Society of Toxicology in Szczyrk in 2008. In contrast to the authors of the commented article we used immunoradiometric assay (IRMA KIT IMMUNOTECH), routinely used for in vitro determination of insulin in human serum and plasma. This method is very sensitive, specific and – what is important – relatively cheap,

Morphological and enzymatic changes caused by a long-term treatment of female rats with a low dose of gonadoliberin agonist and antagonist

Summary Background Long-term treatment with gonadoliberin analogs is used to block the hypothalamic-pituitary-gonadal axis. The use of these agents is generally considered to be safe; however, some observations suggest the possibility of adverse effects. Material/Methods We investigated whether a 3-months administration of a low dose (6 μg/kg b.w.) of dalarelin – a new agonist, and cetrorelix – a known antagonist of GnRH to female rats causes morphological changes in pituitary gland, ovaries, uterus and liver (HE and VG staining); effects on pituitary, hepatic and blood enzyme activities (histochemical and kinetic methods, respectively), and on the blood lipid profile (colorimetric methods); and to what extent these changes are reversible. Results Applying analogs effectively inhibited ovulation, affected the uterine endometrium and changed histological appearance of the liver (e.g., steatosis). They altered activities of marker enzymes of cellular respiration, gluconeogenesis and intracellular digestion in the liver and, partially in the pituitary gland, caused undesirable changes in the activities of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and creatine kinase, and a concentration of cholesterol HDL fraction and triglycerides in the blood. Both morphological and enzymatic effects were more evident after antagonist administration; changes in the blood lipid profile were more evident after agonist administration. In both analogs histological and enzymatic changes persisted a relatively long time after the discontinuation of the treatment. Conclusions The low dose of dalarelin and cetrorelix is sufficient to cause limited damage of hepatic cells and may modify the function of pituitary, ovaries, uterus and liver as well as other organs, even after discontinuation of the treatment.

Transcription Factors Potentially Involved in Regulation of Cytochrome P450 Gene Expression

Drug-metabolizing enzymes, including the cytochrome P450 (CYP) superfamily of enzymes, are subject to regulation by both exoand endogenous factors, mostly hormones and cytokines (Monostory et al., 2009; Waxman & Chang, 2005). In this regulation transcription factors are the mediators. Among them, orphan nuclear receptors: CAR (Constitutive Androstane Receptor), PXR (Pregnane X Receptor), VDR (Vitamin D Receptor), FXR (Farnesoid X Receptor), LXR (Liver X Receptor), PPAR┙ (Peroxisome Proliferator-Activated Receptor α) and RXR (Retinoid X Receptor) are the most important. They can create heterodimers in any configuration what, in conjunction with a broad spectrum of attached ligands, reflects the complexity of regulatory networks (Honkakoski & Negishi, 2000; Xu et al., 2005). Expression of some CYP isoforms is dependent on gender, which partly explains the metabolic difference between men and women in pharmacokinetics of drugs or, for instance, in susceptibility to carcinogens (Scandlyn et al., 2008). The main role in the sex-dependent regulation of CYP expression plays the growth hormone (GH) and to a lesser extent – other hormones. In principle, there are significant differences between genders in the daily profile of GH secretion into the bloodstream (Waxman & Chang, 2005). GH activates signaling pathway JAK-STAT (Lobie & Waxman, 2003). The main regulator of hepatic gene expression dependent on GH is transcription factor STAT5b which, together with other coregulators (i.e. HNF-4┙) can stimulate CYP genes directly by the binding to promoter sequences of target genes or indirectly by the activation of gene expressions of the genderspecific transcription factors (Park et al., 2006). As a result, in transactivation of cytochrome P450 genes, we can distinguish at least two pathways: (1) metabolic, dependent on the type of xenoor endobiotic, mediated by several nuclear receptors and (2) signaling, associated with activation of numerous GH-dependent transcription factors. Therefore, some endocrine disorders may cause changes in the drug metabolism, as well as in the CYP-dependent metabolism of endogenous substrates.

Expression of cytochrome P450 2C and 3A in female rat liver after long-term administration of gonadoliberin analogs.

OBJECTIVES Gonadoliberin (GnRH) analogs may be expected to indirectly modify growth hormone (GH) total concentration and its 24-h secretion profile. As a consequence, changes in the levels of GH may modify the mechanism of sex-dependent cytochromes P450 (CYP450) synthesis, including the expression of transcriptional factors. The aim of the study has been to evaluate the effect of long-term administration of a low dose of GnRH analogs on hepatic expression of CYP2C and CYP3A isoforms, and the transcription factors: pregnane X receptor (PXR), hepatocyte nuclear factor 4α (HNF4α), HNF6 and signal transducers and activators of transcription 5b (STAT5b). MATERIAL AND METHODS The study was carried out on adult female Sprague-Dawley rats during a 3-month treatment with dalarelin (GnRH agonist) and cetrorelix (GnRH antagonist), at a daily intraperitoneal injection (i.p.) dose of 6 μg/kg body weight/day, and 1, 2, and 4 weeks after treatment discontinuation. The concentrations of ovarian hormones and GH in the blood serum were determined by radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) method, respectively. Then, the expression of hepatic CYP450s (reverse transcription polymerase chain reaction - RT-PCR, Western blot and immunohistochemistry) and transcription factors (RT-PCR) was evaluated. RESULTS We have found that cetrorelix induces changes in the circadian pattern of GH secretion and enhances GH blood concentrations. These changes may cause increased expression of both, female-specific CYP450s (especially CYP3A9), and HNF4α/HNF6 transcription factors. Decrease in GH blood concentrations, resulting from the effect of dalarelin, may promote inhibition of female-specific CYP2C12 and CYP3A9 isoforms as well as STAT5b transcription factor. Slight changes in sex-independent CYP3A1 protein expression caused by GnRH analogs were also observed. CONCLUSIONS In adult female rats, HNF4α/HNF6 and STAT5b seem to be crucial for the regulation of GnRH antagonist/GH- and GnRH agonist/GH-dependent pattern of CYP450 expression, respectively.

Evisceration and intestine damage as a rare form of suicide in forensic and psychiatric practice – description of two cases

Suicide is a major public health problem around the world. Case reports include extreme suicidal self-inflicted injuries, in which extensive damage to the abdominal wall associated with injury or even excision of fragments of the intestine and its fragmentation are present. These cases usually give rise to doubts of investigators as to the course of the incident, the circumstances of death and the possibility of participation of other people. At the same time they are interesting from the medico-legal and psychiatric perspective. The aim of this study is the presentation of two extremely rare cases of suicides through evisceration and intestinal injury from the clinical and opinioning practice of the authors, one of which ended with survival and the second one with death. Regardless of the final result of the suicide attempt (death or survival), good practice of the investigative teams in such cases should include a detailed examination of the place of the suicide attempt, obtaining opinion of an expert in the field of forensic medicine with full post-mortem diagnosis, and in-depth forensic psychological and psychiatric analysis of, among others, lifeline, mental state and suicidal motivation (so-called psychological autopsy).

Patient’s death as a result of mistakenly injection of pancuronium

Pancuronium is a typical non-depolarizing, curare-mimetic, very potent muscle relaxant. Besides application in anesthesiology and intensive care, it is used in execution as a part of lethal injection. In medico-legal practice, there are cases of using this substance in order to commit suicide or to deprive other people of their lives. Accidental pancuronium intoxications are very rare. The authors present such case ended in sudden death of hospitalized woman after mistakenly injection of the drug. 57-year-old female alcoholic was admitted to the Acute Poisoning Centre after ethylene glycol ingestion. During the fifth day of treatment the nurse by mistake, instead of furosemide, intravenously administered her pancuronium. Sudden respiratory and circulatory arrest occurred, so she was intubated and resuscitation with artificial ventilation were undertaken, however within 1 hour and 45 minutes the patient died. Due to the vague background of a sudden deterioration in the patients condition, the case was brought for prosecution. The autopsy and histopathological studies did not reveal the cause of death, but undertaken chemico-toxicological examinations identified the presence of pancuronium in blood, liver and kidney (190 ng/ml, 70 ng/g and 125 ng/g, respectively). Chemico-toxicological analysis proved that the cause of death of the 57-year-old hospitalized woman was pancuronium intoxication due to evident medical error during drug administration. In our case the concentration of pancuronium in blood was in therapeutic range (200-600 ng/ml). However, even a therapeutic pancuronium dose administered to patient the breath of whom is not supported and monitored can be a threat to his life.