Acute high-dose diazepam reduces total bilirubin in bile: an experimental mouse study

Drug-related morbidity and mortality have increased worldwide, with mental and substance use disorders representing a substantial component of the global burden of disease [1, 2]. Benzodiazepines are widely prescribed, and their involvement in both accidental and intentional overdoses has been reported in several countries [3, 4]. In forensic practice, benzodiazepine intoxication is typically diagnosed by integrating autopsy findings with postmortem toxicology, although interpretation often relies heavily on measured drug concentrations.

Conventional postmortem toxicology primarily relies on blood and urine, and conclusions are frequently drawn by comparing measured concentrations with reference ranges or reported lethal levels [5]. This approach can be problematic for rare compounds, novel psychoactive substances, or drugs with pronounced postmortem redistribution, and several case reports have highlighted difficulties in assigning cause of death when concentration data are limited or ambiguous [6‐8]. There is therefore a need for complementary indicators that reflect toxicodynamic or pathophysiological changes rather than drug levels alone [9, 10].

Bile represents an attractive alternative matrix in forensic toxicology because it may contain higher concentrations of certain drugs and metabolites than blood, and it is relatively well preserved in decomposed bodies [11, 12]. At the same time, bile composition is influenced by hepatic transport and excretory mechanisms, and interpretation of postmortem bile drug concentrations remains challenging [13, 14]. Bilirubin is a canonical hepatobiliary analyte produced by heme catabolism, conjugated with glucuronic acid in hepatocytes, and excreted into bile [15]. Because glucuronidation is shared between bilirubin and many drugs, intensive drug metabolism could transiently alter bilirubin handling [16].

Diazepam is a long-acting benzodiazepine and GABA_A receptor agonist that undergoes extensive oxidative metabolism followed by glucuronidation [17, 18]. At high doses, diazepam produces sedation, muscle relaxation, and dose-dependent cardiorespiratory effects in experimental animals [19, 20]. We hypothesized that acute, high-dose diazepam administration may transiently perturb hepatic conjugation capacity and thereby modify biliary bilirubin content, even in the absence of overt hepatocellular injury or cholestasis.

This study therefore examines changes in total bilirubin in serum and bile, together with vital signs, in a mouse model of acute high-dose diazepam exposure. Our aim is to explore whether biliary bilirubin could serve as a candidate marker of altered hepatic conjugation during acute benzodiazepine intoxication and to provide a basis for future translational studies in forensic settings.

This study describes an experimental mouse model in which an oral dose of diazepam close to the reported LD₅₀ produced marked bradycardia and sedation without lethality or overt cholestasis, yet was associated with a significant reduction in biliary total bilirubin. This pattern is compatible with a specific alteration in hepatic conjugation and excretory processes during acute diazepam exposure, although the present study did not directly assess hepatic histology or global liver function.

One plausible mechanistic explanation is competition for hepatic glucuronidation capacity between diazepam (and its metabolites) and bilirubin. Bilirubin is conjugated predominantly by UDP-glucuronosyltransferase 1A1 (UGT1A1), and disturbances in this pathway can alter biliary bilirubin excretion without immediate changes in serum levels [15]. Many drugs, including benzodiazepines, undergo glucuronidation by overlapping UGT isoforms [16, 18]. Experimental and clinical data indicate that high substrate loads or co-administered drugs can saturate or competitively inhibit hepatic glucuronidation, leading to altered conjugation of endogenous substrates such as bilirubin [9]. In the present study, biliary total bilirubin decreased in diazepam-treated mice while serum bilirubin remained below the limit of quantification. A reduction in biliary bilirubin without detectable accumulation in serum suggests that conjugated bilirubin formation or canalicular transport may have been transiently constrained, but not to a degree sufficient to produce systemic hyperbilirubinemia. This pattern is compatible with the hypothesis that limited hepatic conjugation capacity was preferentially utilized for diazepam and its metabolites rather than for bilirubin. However, this mechanism should be regarded as diazepam-specific until comparable data are available for other GABA_A receptor agonists.

The vital sign changes observed in this study are consistent with the known pharmacological profile of high-dose diazepam. Preclinical studies have reported dose-dependent reductions in locomotor activity and cardiovascular depression following large diazepam doses in mice [19, 20]. Clinical literature on benzodiazepine poisoning similarly describes bradycardia and hypotension, particularly in severe cases or when combined with other central nervous system depressants [23, 24]. In the present model, diazepam produced a sustained decrease in pulse rate without major hypoxemia or respiratory depression (Fig. 1a–c), indicating a pharmacologically effective but non-lethal exposure. This context supports the interpretation that the observed reduction in biliary bilirubin occurred prior to the development of severe cardiorespiratory compromise.

Bile is increasingly recognized as a valuable but underused matrix in forensic toxicology, particularly in decomposed bodies or when blood is unavailable [11‐14]. However, the toxicological significance of bile drug concentrations is difficult to interpret without reference to underlying hepatobiliary physiology. The present findings suggest that biliary bilirubin content may provide additional information on hepatic conjugation status in the setting of acute diazepam intoxication. Importantly, because bilirubin conjugation and biliary excretion can be altered by many conditions and xenobiotics, reduced biliary bilirubin should not be interpreted as a diazepam-specific indicator or a stand-alone basis for attributing exposure. Rather, if similar patterns are observed in human autopsy bile, biliary bilirubin may be considered a supportive, mechanism-oriented finding to be interpreted alongside comprehensive toxicological results, case circumstances, and pathological evaluation. At the same time, any such interpretation must account for pre-existing liver disease, cholestasis, and postmortem changes in bile composition.

This study has several limitations. First, the sample size was modest, and only male ICR mice were examined at a single, very high dose and at a single time point. Second, bilirubin concentrations in serum were below the limit of quantification, and bile volume was limited, which precluded reliable fractionation into conjugated and unconjugated bilirubin or simultaneous measurement of additional biliary constituents. Third, the study did not directly quantify diazepam or its metabolites in bile or liver, and therefore the proposed competition for glucuronidation remains inferential. Finally, extrapolation of these findings to humans or to other benzodiazepines is uncertain and will require systematic analysis of bile and related markers in human forensic autopsy cases, together with further studies in additional experimental models.

Despite these constraints, this model demonstrates that biliary total bilirubin can decrease measurably after acute high-dose diazepam administration in the absence of overt cholestasis or lethal toxicity. Future work should examine dose–response relationships, temporal dynamics, and the influence of underlying liver disease, and should include simultaneous measurement of hepatic and biliary diazepam metabolites. Ultimately, integrating bile-based indices of hepatic conjugation with conventional postmortem toxicology may help refine the interpretation of suspected benzodiazepine intoxication in forensic casework.

Acute high-dose oral diazepam administration in mice produced marked bradycardia with minimal respiratory compromise and a significant reduction in biliary total bilirubin, while bile weight and serum bilirubin remained unchanged. These findings suggest that biliary bilirubin content may serve as a candidate indicator of altered hepatic conjugation during acute diazepam intoxication. Validation in human autopsy samples and across different dosing conditions will be essential before this approach can be adopted as a routine adjunct in forensic toxicology.