Trauma-toxicology: concepts, causes, complications

Trauma and toxic substances are connected in several aspects. On the one hand, toxic substances can be the reason for traumatic injuries in the context of accidental or violent and criminal circumstances. Examples for the first scenario is the release of toxic gases, chemicals, and particles during house fires, and for the second scenario, the use of chemical or biological weapons in the context of terroristic activities. Toxic substances can cause or enhance severe, life-threatening trauma, as described in this review for various chemical warfare, by inducing a tissue trauma accompanied by break down of important barriers in the body, such as the blood-air or the blood-gut barriers. This in turn initiates a “vicious circle” as the contribution of inflammatory responses to the traumatic damage enhances the macro- and micro-barrier breakdown and often results in fatal outcome. The development of sophisticated methods for detection and identification of toxic substances as well as the special treatment of the intoxicated trauma patient is summarized in this review. Moreover, some highly toxic substances, such as the protein toxins from the pathogenic bacterium Clostridioides (C.) difficile, cause severe post-traumatic complications which significantly worsens the outcome of hospitalized patients, in particular in multiply injured trauma patients. Therefore, novel pharmacological options for the treatment of such patients are necessarily needed and one promising strategy might be the neutralization of the toxins that cause the disease. This review summarizes recent findings on the molecular and cellular mechanisms of toxic chemicals and bacterial toxins that contribute to barrier breakdown in the human body as wells pharmacological options for treatment, in particular in the context of intoxicated trauma patients. “trauma-toxicology” comprises concepts regrading basic research, development of novel pharmacological/therapeutic options and clinical aspects in the complex interplay and “vicious circle” of severe tissue trauma, barrier breakdown, pathogen and toxin exposure, tissue damage, and subsequent clinical complications.


Introduction
Physical trauma is the realization of an external danger impact which threatens any life at any time.In humans and animals, the physical trauma force vector can breach the protective layers encompassing of the skin, fasciae, capsules, and underlying tissues.This damage results in the generation and release of damage-associated molecular patterns (DAMPs) including membrane debris, mitochondrial components, histones, DNA-and RNA fragments, and damaged proteins (Huber-Lang et al. 2018) (see Fig. 1).The demolished and compromised barriers inadequately restrict the efflux of vital internal constituents, such as blood, into the external environment, leading to circulatory disturbances, hypoperfusion, hypoxia, and shock.Conversely, these compromised external 1 3 and internal barriers become highly susceptible to the ingress of microorganisms and potentially hazardous substances and fluids (Huber-Lang et al. 2018), thus increasing the risk of infectious, toxic, or septic complications.Recent findings from our laboratory have demonstrated that a highly standardized murine polytrauma plus hemorrhagic shock results in remote intestinal injury characterized by an enhanced permeability of the gut-blood-barrier (GBB) (Wrba et al. 2019), which can ultimately impair organ performance and culminate in the development of multiple organ dysfunction syndrome (MODS), often resulting in a fatal outcome (Huber-Lang et al. 2018).An effective yet limited sealing system of the disrupted tissues is the fluid-phase and cellular coagulation system, which rapidly activates in the aftermath of trauma to prevent leakage (Rossaint et al. 2023).Similarly, activation of the interconnected innate complement cascade (Burk et al. 2012) and the "first cellular line of defense" strive to repel microbial invasion and to inhibit their growth.
Trauma as a physical "wound" can manifest across various contexts, including civilian incidents, acts of terrorism or within military operations.It encompasses physical injuries, burns, as well as exposure to chemical, biological, or radiological/nuclear (CBRN) warfare agents.While bioterrorism and B-warfare-so far known-predominantly involve agents such as viruses, bacteria, and bacterial toxins (Botulinum Neurotoxin (Kreyden et al. 2000), Anthrax toxin) as well as plant toxins (Ricin) (Tin et al. 2022), the arsenal of chemical warfare agents is extensive (Wille et al. 2011) and comprises toxic substances that affect the skin, the lungs, or the nervous system.The latter comprises organophosphates such as tabun, sarin, VX, and the Novichok compounds, which can cause a severe, life-threatening cholinergic syndrome, as described in more detail later.These agents possess nearly boundless potential for inflicting tissue damage.
Furthermore, combinations of traumata in the spatio-temporal dimension, i.e., simultaneously across different body regions or consecutive impacts, can aggravate the overall trauma load.As a side note, the concept of trauma extends beyond physical injuries to encompass psychosocial dimensions.Research has unveiled a growing understanding of the intricate physio-psychological interactions associated with trauma (Haffner-Luntzer et al. 2019).Notably, psychological stress has been shown to significantly alter the composition of the gut microbiome, potentially influencing the presence of exo-and endotoxin-producing microbes (Langgartner et al. 2018).However, the focus of this review does not encompass psychological aspects of trauma, which remain beyond the scope of our discussion.

Chemical warfare agents
Chemical warfare represents a dark chapter in the history of armed conflicts, encompassing the deliberate use of chemical substances to inflict harm upon adversaries.Unlike conventional weapons, which rely on explosives or projectiles, chemical weapons leverage the toxic properties of chemicals to cause injury, incapacitation, or death.Natural toxins from plants or animals can be regarded as the earliest types of chemical warfare agents (CWAs).One well-known example is curare, a poison derived from certain plants found in South America that works by blocking neuromuscular transmission, leading to muscle paralysis and ultimately respiratory failure (Bowman 2006).
World War I (WWI, 1914 to 1918) stands as a pivotal moment in the history of warfare: The first large-scale use of chlorine gas from pressurized canisters across a 5-mi front by the German forces, engulfing Allied positions, has to be regarded as "zero hour" of chemical warfare in modern history (Black 2016).During WWI, the deployment of CWAs in massive quantities (about 125,000 tons), resulted in about 90,000 fatalities and 1.3 million nonfatal casualties.The use, large-scale development, production, stockpiling, or transfer of chemical weapons or their precursors is meanwhile prohibited by the Chemical Weapons Convention that entered into force in 1997.Nonetheless, chemical weapons have been utilized in a number of subsequent conflicts, terrorist attacks, or assassinations (Ganesan et al. 2010).The most recent incidents include the murder of Kim Jong-nam (VX) and the attempted killings of Sergei Skripal and Alexei Navalny (both with Novichok) (Brunka et al. 2022).

Classes of chemical agents
Chemical warfare agents are categorized into several groups based on their chemical properties, mechanisms of action, and pathophysiological effects.The most common classification system divides them into six categories (see Table 1).Blister agents cause severe skin, eye, and mucous membrane damage.They are named for the large, painful blisters that they can cause.Blood agents are compounds that interfere with the body's ability to utilize oxygen.They are typically absorbed through the respiratory system but can also be swallowed.Examples include hydrogen cyanide (AC) and cyanogen chloride (CK).Pulmonary agents damage lung tissue, often leading to suffocation.Examples include chlorine (Cl), phosgene (CG), and chloropicrin (PS).Nerve agents disrupt the neuronal signal transmission, leading to cholinergic overstimulation.Examples include sarin (GB), soman (GD), tabun (GA), VX, and the Novichoks.
In addition to these, there are also riot control agents, which are chemicals used for law enforcement and crowd control.These chemicals, while not intended for lethal use, can cause immediate irritation of the eyes, nose, and respiratory tract.Mental incapacitating agents include drugs and compounds that affect functions of the central nervous system resulting in respiratory depression and loss of consciousness.Examples are fentanyl and derivatives thereof.Because these compounds are used as drugs in clinical routine, they are also referred to as "pharmacological-based agents."Furthermore, some chemical agents may exhibit characteristics of multiple categories.For example, certain compounds can act as both blister agents and pulmonary agents, depending on the route of exposure.
Chemical warfare compounds have the potential to impair the exposed person's health instantly or within a few hours after exposure.Trauma damage sustained during combat or due to the explosive deployment of the warfare agent may exacerbate the health condition.As a result, the intoxicated trauma patient requires both trauma care and additional treatment due to chemical agent contamination.

The intoxicated trauma patient
The management of this specific group of patients is difficult, yet it adheres to strict guidelines (Wille et al. 2019).
First, early antidote treatment (e.g., atropine and obidoxime in the event of nerve agent poisoning (Amend et al. 2020) or naloxone in the case of opioid is life-saving and should be initiated as soon as possible.The "time-until-first-treatment" is critical and should be kept as short as feasible.Antidotes can be delivered using auto-injectors while still in the hot zone and prior to decontamination.The "trigger-totreat" is usually the manifestation of characteristic chemical agent-related clinical symptoms.However, particularly in the event of percutaneous exposure to non-volatile nerve agents (i.e., VX or Novichoks), symptoms may not manifest immediately, despite the fact that the patient has already been severely poisoned.
Onsite testing of acetylcholinesterase activity using a mobile test kit and the use of a sensitive and easy to use organophosphate (OP) skin disclosure kit may allow for the early diagnosis of OP skin exposure and the start of lifesaving countermeasures (Worek et al. 2016).Contaminated patients cannot be sent into the rescue chain without prior decontamination.Massive bleedings and trauma injuries must also be handled prior to decontamination, for example, using tourniquets, to minimize any treatment delays.In a mass intoxication scenario, the number of patients will exceed the available resources (Rossaint et al. 2023).Thus, there is an urgent need to prioritize equipment and medical supplies using triage algorithms (Khoshnevis et al. 2015).It is critical to repeat the triage procedure on a regular basis in order to detect deteriorating health issues, particularly following percutaneous exposures.

Verification of exposures to chemical warfare agents
Most chemical agents are highly reactive.They readily hydrolyze following either an enzymatic or, more typically, a non-enzymatic pathway.Moreover, they can form covalent products (adducts) with endogenous biomacromolecules (e.g., DNA and proteins) (John & Thiermann 2021).The limited stability and high reactivity of most chemical agents preclude the detection of the intact poison in vivo, thus requiring the search for more stable and long-lived surrogate parameters derived from biotransformation (John et al. 2018).Thus, detection of the metabolites or the adducts can be used to verify human exposures.Gas and liquid chromatography (LC) coupled to mass spectrometry (MS) are commonly used for biomedical verification of OPNA (organophosphorus nerve agent) exposure in humans and animals (Kranawetvogl et al. 2023).

Barrier failure due to trauma
An intact intestinal barrier serves as a crucial boundary separating the systemic circulation from the intestinal microbiome.When this barrier is compromised, it can lead to the translocation of bacteria and pathogen-associated molecular patterns (PAMPs) into the bloodstream or lymphatic system, thereby intensifying the immune response and promoting systemic inflammation (Wrba et al. 2017).This phenomenon is commonly observed as a complication following injury, with profound systemic effects on both pro-inflammatory and anti-inflammatory immune responses, as well as organ perfusion and oxygenation.Such intestinal barrier dysfunction often contributes to multiple organ dysfunction in the clinical course post-injury (Faries et al. 1998;Spindler-Vesel et al. 2006).In case of direct abdominal trauma, experimental evidence suggests, that the infliction of macroscopically visible intestinal injuries is significantly influenced by the location and intensity of the traumatic force (Maitz et al. 2021).In the clinical setting, indirect, remote abdominal traumata appear more frequent than direct ones.Especially, severe blood loss with development of hemorrhagic shock during or after traumatic injury emerges as a major factor causing endothelial damage and subsequent dysfunction of the intestinal barrier (see Fig. 2).
Our own research findings further emphasize the vulnerability of the intestines to hypoperfusion due to blood loss or centralization.In patients with multiple injuries, we observed a remarkable increase in the levels of circulating glycocalyx components and markers of intestinal injury and permeability, particularly in those who had experienced substantial blood loss (Halbgebauer et al. 2018).In a murine model of multiple trauma and hemorrhage, we observed several notable effects on intestinal integrity even in the absence of direct abdominal injury.These effects included an increase in abdominal girth, indicative of extravascular fluid accumulation, a reduction in the expression of the central tight-junction protein, zonula occludens protein 1, in cell-cell contacts within the ileum and colon, and the appearance of mucosal molecules in the bloodstream (Wrba et al. 2019).
These findings underscore the significant impact of systemic post-traumatic processes on the integrity of the intestines.Additionally, it is worth noting that traumatic brain injury, as demonstrated in various preclinical models, can additionally impair intestinal barrier function (Bansal et al. 2010;Feighery et al. 2008;Ma et al. 2019).Mechanistically, we have identified the local upregulation of an apoptosisinducing protein, thirty-eight-negative kinase 1, as a potential mediator of post-traumatic intestinal epithelial cell death (Armacki et al. 2018).For future therapeutic applications, preventing the effects of mesenteric "lymph toxicity" (Magnotti et al. 1998;Deitch et al. 2006;Fang et al. 2010;Levy et al. 2013) may aid in reducing the detrimental effects of intestinal injury on remote organ systems.Taken together, the available data underscores the significance of the intestine as an often-underappreciated contributor to the development of post-traumatic complications.

Trauma-caused microbiome shifts: role of bacterial enterotoxins
The human as "macrocosm" and the integrated and surrounding microorganisms as "microcosm" share a complex and mutually dependent relationship.In the experimental setting of rodent polytrauma, both, others, and our research efforts have uncovered rapid microbial alterations within the gut mere hours after injury, a phenomenon influenced, among other factors, by the systemic inflammatory response and the demand for catecholamines (Nicholson et al. 2018;Appiah et al. 2021).In the days following trauma, a significant shift in the microbiome is observed, characterized by a transition towards a pathobiome.This shift manifests as a loss of beta-diversity and the prevalence of certain microorganisms such as Rothia, Anaerostipes, and Lactobacillus.Simultaneously, indications of a compromised intestinal barrier emerge, as recently demonstrated in a rat model of multiple injuries and secondary insults (Munley et al. 2023).Studies employing rodent burn injury revealed that advanced age exacerbates microbiome dysbiosis and weakens the host's antimicrobial defenses (Wheatley et al. 2020).Conversely, adolescent rats subjected to repetitive mild traumatic brain injury (TBI), preceded by microbiome depletion, exhibited a pathogenic state dominated by Clostridia, rendering them more vulnerable than their adult counterparts (Sgro et al. 2022).
Translational research has consistently revealed that microbial changes are closely associated with adverse outcomes (Schuijt et al. 2013).Several factors, notably traumainduced hypoxia, stress, and administration of antibacterial drugs, have been identified as key drivers of microbiome alterations.In patients with persistent hypoxia (paO 2 /FiO 2 ratio below 300) following burn and inhalation injuries, an altered bronchoalveolar microbiome is evident, characterized by an enrichment of Prevotella, Corynebacteria, and Mogibacterium (Walsh et al. 2017).Likewise, spinal cord injury studies, both experimental and clinical, have identified an increase in inflammation-promoting microbes, including Clostridia (Valido et al. 2022).Despite the growing body of evidence pointing to early post-trauma microbiome alterations, our understanding of the specific pathobiome and the mechanisms involved in rebalancing microbiome homeostasis following trauma remains limited.Further investigations are warranted to unravel these intricate interactions.
Numerous microorganisms possess the capability to facilitate microbial invasion by strategically targeting and compromising the intestinal barrier through the action of enterotoxins.These enterotoxins act predominantly in the gut and mainly target the intestinal epithelial cells, often exhibiting pore-forming properties, or disrupt the integrity of intercellular tight junctions, resulting in a loss of barrier function.Enterotoxins can be produced by a variety of pathogens, including Staphylococcus aureus, Bacillus cereus, and Streptococcus pyogenes, and by plants (Ricin), as summarized in Table 2.
Notably, enterotoxins, especially the AB-toxins produced by C. difficile (Aktories 2011) and Clostridium perfringens, play a significant role in this context.In serum and wounds of burn and trauma patients, enterotoxin A from Staphylococcus aureus (SEA) could be isolated and were predictive of mortality (Ali et al. 2022;Prindeze et al. 2014).In a rat model of infectious burn wounds, enterotoxin B (SEB) and toxic shock syndrome toxin-1 (TSST-1) were found to translocate from the wounds to the kidneys, potentially contributing to the development of remote complications (Mino et al. 2013).Collectively, research on enterotoxins has predominantly centered on burn injuries, necessitating further comprehensive mechanistic investigations in diverse trauma and post-traumatic settings.

Role of bacterial toxins in traumatic diseases, barrier failure, and post-traumatic complications
Nowadays, due to changing global political situations, there is increasing concern regarding the deployment of C-and B-weapons in the context of military or terroristic activities, also in Europe.Therefore, research on the mode of action of such toxins is of major impact, as well as on the development of novel, highly specific and sensitive detection methods for such compounds and therapeutic options to treat traumatic and post-traumatic diseases caused by C-and B-warfare.
We and others have investigated the mode of action of the protein exotoxins of C. difficile on the molecular and cellular levels in detail and suggested a panel of molecules including human body's own proteins and peptides as well as licensed drugs that are used for therapy of other diseases as potent inhibitors against C. difficile toxins.These molecules, in addition to the already available antibacterial drugs against C. difficile, might provide an attractive starting point for clinical studies to introduce novel pharmacological options to treat and/or prevent C. difficile-induced infections (CDI).
Similar to the trauma response, the response to toxic substances including bacterial protein toxins can result in local and systemic inflammation, activation and depletion of the coagulation-and complement cascade, development of barrier dysfunction, micro-and macro-perfusion problems, and subsequent organ dysfunction and failure (Abrams et al. 2022).Although the clinical consequences of toxins can be similar, toxic compounds and their induced pathophysiology can be rather different.In conclusion, toxic compounds exhibit a wide range of characteristics, yet their clinical manifestations can resemble those of other severe diseases, potentially leading to multiple organ failure.However, little is known about the role of toxins in the context of severe tissue trauma and the potential underlying crosstalk driving the pathophysiology towards disease progress including the process of post-traumatic regeneration.In particular, barrier failure due to toxic compounds such as chemical warfare or toxins (e.g., enterotoxins from C. difficile) needs further mechanistic enlightenment.

Trauma-associated Clostridioides difficile infection
In recent decades, there has been a global increase in Clostridioides (formerly Clostridium) difficile infections, impacting not only the traditional demographic of elderly patients with prolonged use of antibacterial drugs (Depestel & Aronoff 2013).In case of accidental or surgical trauma, C. difficile infection develop in ca.1-3% (Gonzalez et al. 2022;Lumpkins et al. 2008).Moreover, in cases of burn injuries, colonization by C. difficile on the skin and in the surrounding environment has been reported in up to 18% of cases (Shoaei et al. 2022).An extensive analysis of over 11,000 trauma patients admitted to a level I trauma center revealed a significant association between C. difficile infection and a nearly threefold increase in mechanical ventilation requirements, mortality rates, and a markedly prolonged stay in both the intensive care unit (ICU) and overall hospitalization duration (Karamanos et al. 2018).
Patients with post-traumatic C. difficile often exhibited a high initial injury severity score (ISS), an abdominal injury pattern (colonic, renal, and hepatic), received third-generation cephalosporins and/or clindamycin, and/or i.v.proton-pumpinhibitors (Karamanos et al. 2018).In addition, a clinical analysis after blunt trauma proposed a specific C. difficile patient population that was older than 65 years, and developed greater multiple organ dysfunction scores (including enhanced base deficit, lactic acid, creatinine, glucose levels, and reduced PiO 2 :FiO 2 ) than uninfected trauma patients (Vanzant et al. 2015).A recent multifactorial analysis reaffirmed these risk factors and introduced trauma as a surgical cause associated with C. difficile infection (Jachowicz et al. 2022).
It is noteworthy that an analysis of approximately 1.5 million surgical patients indicated that an elevated body mass index, a measure of adiposity, appears to confer some protection against C. difficile infection (Meier et al. 2019).Recognizing the challenge of C. difficile infection in trauma patients, management guidelines have been developed to address this concern (Sartelli et al. 2019(Sartelli et al. , 2021)).Therapeutically, application of phosphatidylcholine as a key component of the intestinal mucosal barrier reduced epithelial necrosis and improved the barrier integrity in an in vitro intestinal model of C. difficile exposure (Olson et al. 2014).However, translating these findings into clinical interventions for restoring the gut-blood barrier in cases of C. difficile infection remains a pending challenge.

Clostridioides difficile toxins as reason for post-traumatic complications
Structure, uptake, mode of action, and pathophysiological role of TcdA and TcdB Toxin A (TcdA) and toxin B (TcdB) are the two major exotoxins of C. difficile (Aktories 2011;Just et al. 1995a, b).A third toxin termed CDT (C.difficile transferase) is produced by certain epidemic strains (e.g., C. difficile strain BI/ NAP1/027).All three toxins are protein toxins harboring a deleterious enzymatic domain, which is delivered into target cells via receptor-mediated endocytosis.The toxins' actions on target tissues are directly responsible for the outcomes or severity of C. difficile-associated diseases (CDADs), such as diarrhea and pseudomembranous colitis (Papatheodorou et al. 2018).
TcdA and TcdB (shortly TcdA/B) are independently acting, single-chain toxins with a rather large size of 308 (TcdA) and 270 kD (TcdB), respectively.They consist of several functional domains whose orchestrated interplay is required for the delivery of the toxic cargo, an N-terminally located glucosyltransferase domain, into target cells (Aktories et al. 2017).The domain architecture of TcdA/B and their functions at consecutive steps during cell entry of TcdA/B are depicted in detail in Fig. 3A.
The target molecules of TcdA/B are small GTPases of the Rho and/or Ras family (Just et al. 1995a, b;Just & Gerhard 2004;Genth et al. 2018;Zeiser et al. 2013), which act in cells as molecular switches and as master regulators of the actin cytoskeleton and of numerous other cellular processes, including cell migration, phagocytosis, intracellular trafficking, cell progression, and apoptosis (Nobes & Hall 1994;Burridge and Wennerberg 2004 l;Jaffe & Hall 2005;Aktories 2011;Lemichez & Aktories 2013).TcdA and TcdB vary in their substrate profile but they both inactivate their substrates by covalent attachment of a glucose moiety at a conserved threonine residue, which is crucial for the interaction with effectors.The glucose is provided by UDP-glucose, which acts as a co-substrate for the toxins' glucosyltransferase domain.In this respect, TcdA/B are members of the family of clostridial glucosylating toxins (CGTs) (Jank & Aktories 2008), also denoted as the family of large clostridial cytotoxins (LCCs) (von Eichel-Streiber et al. 1996).
TcdA/B-mediated glucosylation of Rho proteins causes a number of changes in cellular function, but cell rounding is the most obvious cytopathological effect in cultured mammalian cells.Due to a redistribution of the actin cytoskeleton, the cells lose their normal shape and form irregular extensions, a process which is called arborization.The role of the toxins during CDI pathogenesis is not entirely understood.However, pathophysiological effects of the toxins, such as disruption of the barrier function of enterocytes, impairing colonic epithelial renewal, increasing colonic vascular permeability, induction of apoptosis, and pro-inflammatory activities, are altogether contributing and promoting disease pathogenesis.The numerous cytopathological and pathophysiological effects of TcdA/B were summarized recently in detail (Papatheodorou et al. 2018).

Structure, uptake, mode of action, and pathophysiological role of CDT
CDT is binary toxin formed by two separate components, the binding and translocation component CDTb and the enzyme component CDTa.The toxin is highly similar to other clostridial binary ADP-ribosylating toxins, such as the C2 toxin from Clostridium botulinum and the iota-toxin from Clostridium perfringens, and more distantly related to the anthrax toxin of Bacillus anthracis (Stiles et al. 2014;Aktories et al. 2018).CDTb binds to its receptor, the lipolysis-stimulated lipoprotein receptor (LSR) (Papatheodorou et al. 2011), and upon entry into endosomes, and facilitates by forming oligomeric pores the translocation of CDTa into the cytosol.In the cytosol, CDTa utilizes NAD for covalent attachment of an ADP-ribose moiety to monomeric G-actin.ADP-ribosylated G-actin monomers block the polymerization of F-actin filaments, which eventually leads to the collapse of the actin cytoskeleton.Figure 3B illustrates the modular composition of CDT and its various steps during cell entry.Recent findings indicate that CDTb alone is also capable of damaging cells by pore formation in the plasma membrane (Landenberger et al. 2021).
The pathophysiological role of CDT still remains enigmatic.However, a recent retrospective study has shown that CDT-positive patients were associated with increased disease severity and worse clinical outcomes (Young et al. 2022).One explanation might be that CDT increases the adherence of the C. difficile bacteria at the surface of intestinal epithelial cells.CDT-induced disruption of the actin cytoskeleton leads to the formation of long microtubulebased protrusions on the surface of intestinal host cells.These protrusions enwrap the bacteria, resulting in increased pathogen adherence (Schwan et al. 2009).

Novel pharmacological approaches against C. difficile toxins
The growing mechanistic knowledge about the biology of C. difficile toxins has led to novel anti-toxin approaches, which might be useful in the future as supportive treatment options against C. difficile-associated diseases and/or posttraumatic complications.For instance, body-own antimicrobial peptides, such as certain defensins, were shown to inhibit TcdA/B and CDT either by direct interaction and formation of biologically inactive aggregates (Fischer et al. 2020;Korbmacher et al. 2020;Barthold et al. 2022) or by inhibiting enzyme activities, as suggested for human α-defensin and TcdB (Giesemann et al. 2008).
Along with body-own or artificial peptide libraries, "drug repurposing" (also known as "drug repositioning") is another promising approach for the discovery of novel pharmacological approaches against C. difficile toxins on the basis of already licensed and safe-to-use drugs.It is a cost-effective and time-efficient way to develop new antitoxin treatments with a high probability of success.
Since now, several licensed drugs have been found that exhibits activity against C. difficile toxins.The mucolytic Pore formation of TcdA/B is another critical step during cell entry and requires the presence of cholesterol in endosomal membranes.Therefore, the hypocholesterolemic drug simvastatin, which acts as an inhibitor of the HMG-CoA reductase, was found to be capable of inhibiting cell entry of TcdA/B by decreasing the cholesterol content in membranes of cultured cells (Papatheodorou et al. 2019).Recently, it was shown that the antiarrhythmic drug amiodarone prevents intoxication of cells by TcdA/B (Schumacher et al. 2023).Amiodarone's main mode of inhibition likely involves interference with pore formation and translocation of both toxins.
For CDT, not only domperidone, but also the antibacterial drug bacitracin and the immunosuppressant cyclosporine A have been proven to interfere with the endosome-to-cytosol translocation of CDTa and to thus inhibit the intoxication of cells with CDT (Schnell et al. 2019).Interestingly, later, it was found that bacitracin was also effective against TcdB, most likely due to inhibition of the translocation of the glucosyltransferase domain across the endosomal membrane (Zhu et al. 2019).Another approach for inhibiting CDT is the direct blockage of its pore formed by CDTb with chemical compounds.The antimalarial drug chloroquine is among various substances that are capable of inhibiting the CDTb pore, thereby preventing not only the endosome-to-cytosol translocation of CDTa, but also the cytotoxic effects, which are associated with CDTb-dependent pore formation at the plasma membrane (Ernst et al. 2021).Licensed drugs capable of inhibiting TcdA/B and/or CDT and their proposed mode of inhibition are summarized in Fig. 3C.

Conclusions
Trauma-toxicology, traumatology, and toxicology are related medical fields, but they have distinct focuses and approaches (see Table 4).Trauma-toxicology is a specialized area of toxicology that focuses on the effects of toxic substances on injured patients, while traumatology focuses on the prevention, diagnosis, and treatment of injuries (see Table 3).
In conclusion, for the first time, we coin and define the term "trauma-toxicology" referring the interplay and "vicious circle" of severe tissue trauma, barrier breakdown, pathogen and toxin exposure, and subsequent tissue damage.Furthermore, trauma can be associated with toxins (e.g., biological warfare) and toxic agents (e.g., chemical warfare) leading also to macro-and micro-barrier break down and the "vicious circle" and frequently often fatal outcome.In this context, toxins can be the reason for the trauma or essentially contribute to post-traumatic complications after traumatic injuries.Although the underlying cellular and molecular mechanisms of both toxins and toxic agents are well described for most substances, treatment options need further research and translation to the real world in order to sufficiently improve traumatoxicological conditions.Some keyterms in the field of "trauma-toxicology" are defined in Table 4.

Fig. 1
Fig. 1 The concept of trauma-toxicology: The interplay between trauma and toxic agents and their biological consequences.Explanations are given in the text

Fig. 2
Fig.2The multiple aspects of trauma-toxicology.Explanations re given in the text.CNS, central nerve system; DAMPs, danger-associated molecular pattern; PAMPs, pathogen-associated molecular pattern

Fig. 3
Fig. 3 Domain architecture, cellular uptake, mode of action, and inhibitory drugs of C. difficile toxins.A Domain architecture of TcdA and TcdB.The glucosyltransferase domain (GTD, red) is the toxic part of the toxins that modifies host Rho and/or Ras GTPases upon cleavage and release into the cytosol by the cysteine protease domain (CPD, blue).The combined repetitive oligopeptides (CROPs, gray) act together with the delivery and receptor binding domain (DRBD, yellow) in binding of the toxins to cell surface receptors.The DRBD is also harboring a region that forms a translocation pore in endosomal membranes upon acidification of the endosomes via vacuolar H + -ATPases (shown in brown) for delivery of the GTD into the cytosol.B Domain architecture of CDTa and CDTb.CDTa consists of an N-terminal pADPRT, pseudo-ADP-ribosyltransferase (pADPRT) followed by an active ADP-ribosyltransferase (ADPRT) that modifies G-actin monomers upon entry into the host cell cytosol via CDTb, which consists of the domains D1 (activation by host proteases), D2/ D3 (insertion and pore formation in endosomal membranes upon acidification of the endosomes via vacuolar H + -ATPases (shown in brown) for delivery of CDTa into the cytosol), D3 (oligomerization), and D4 (receptor binding; including a glycan-binding domain (GBD)).C Cellular uptake, mode of action, and inhibitors of TcdB (exemplarily; left part) and CDT (right part).The four steps in Roman numerals indicate I, receptor binding (for CDT receptor binding is

Table 1
Main classes of chemical agents and their mode of action

Table 2
Main toxins involved in traumatic injuries and post-traumatic complications

Table 3
Comparison between traumatology, toxicology, and trauma-toxicology

Table 4
Definition of key terms in trauma-toxicology Term Definition, meaning Pathobiome The microbiome, which underwent a shift towards the growth of more pathogenic bacteria in the gut.This might be induced by prolonged treatment with antibacterial drugs or by traumatic injuries Time until first treatment The time elapsed till the first professional diagnostic measures and treatment is induced Macrocosm The patient Microcosm The microbiome of a patient B-warfare Biological warfare, e.g., viruses, bacteria, toxins from bacteria (anthrax toxins, botulinum neurotoxin) or plants (ricin) C-warfare Chemical warfare (see Table 1) Post-traumatic complications Clinical complications in patients after traumatic injury, such as infections with toxin-producing bacteria in hospital (e.g., C. difficile, Staphylococcus aureus, Streptococcus pyogenes) that may worsen the outcome of these trauma patients Trigger-to-threat Manifestation of characteristic clinical symptoms after exposure to a "trigger" (e.g., a toxic substance) Intoxicated trauma patient A patient that has an intoxication, e.g., by B-or C-warfare in addition to the traumatic injuries C.: Clostridioides; CBRN: Chemical, biological or radiological/nuclear warfare agents; CDAD: C. difficile-Associated disease; CDI: C. difficile-Induced infection; CDT: C. difficile Transferase; ADPRT: CG, phosgene; CGTs: Clostridial glucosylating toxins; CK: Cyanogen chloride (CK); Cl: Chlorine; CNS: Central nerve system; C-warfare: Chemical warfare; CWAs: Chemical warfare agents; DAMPs: Damage-associated molecular patterns; GA: Tabun; GB: Sarin; GBB: Gut-blood-barrier; GD: Soman; GTPases: Guanine nucleotide-binding proteins (G-proteins); ICU: Intensive care unit; ISS: Injury severity score; LC: Liquid chromatography; LCCs: Large clostridial cytotoxins; MODS: Multiple organ dysfunction syndrome; MS: Mass spectrometry; OP: Organophosphate; OPNA: Organophosphorus nerve agent; PAMPs: Pathogenassociated molecular patterns; PS: Chloropicrin; SEA: Enterotoxin A from Staphylococcus aureus; SEB: Enterotoxin B from Staphylococcus aureus; TBI: Mild traumatic brain injury; TcdA: Toxin A from C. difficile; TcdB: Toxin B from C. difficile; TSST-1: Toxic shock syndrome toxin-1; WWI: World War I