\r\n\tUsually, no definite cause can be found for these symptoms. However, it can be secondary to a migraine, ischemic stroke or seizures. It is also commonly seen after minor head injuries, especially amongst athletes. Brain structures responsible for amnesia are temporal lobe regions, especially hippocampal formations. Blood flow studies or cerebral metabolism studies using imaging tests such as positron emission tomography (PET) scan might show hypometabolism in mesial temporal structures. Treatment for amnesia is mainly to prevent recurrence. Finding and managing the cause is the best approach to prevent the relapse of amnesia. We are expecting a range of experts such as psychologist, psychiatrist, neurobiologist, neurologist, and neurosurgeons to submit their chapters in this book.
\r\n\tThis book will not only meant for medical professionals but for the general public who are interested in this topic.
The c-myc oncoprotein is a well-studied, multifunctional transcription factor that controls cell proliferation, metabolism, and stress responses [1, 2]. Deregulation (amplification/overexpression) of c-myc occurs in many human cancers and is considered a “transition gatekeeper” for tumorigenesis. c-Myc controls metabolic reprogramming, a key event in tumorigenesis that provides gain-of-survival adaptive advantage, enabling cancers to thrive in tumor microenvironments with limited nutrient resources [3, 4, 5]. This rewiring of metabolic circuitry is characterized by (i) preferred use of glucose to produce ATP by aerobic glycolysis combined with increased lactate production even in oxygen abundance and (ii) dependence on glutamine for conversion to glutamate and subsequent entry into the tricarboxylic acid cycle by anaplerosis [6, 7]. The metabolic calibration by c-myc in cancer cells is orchestrated to adequately meet the demands of energy expenditure, macromolecular syntheses, and disposal of catabolic wastes, in concordance with robust cell growth. For example, the capacity for aerobic glycolysis in highly proliferative cancer cells is controlled by c-myc via an increase in glucose uptake as a result of induced expression of glucose transporter 1 (Glut1) and lactate dehydrogenase (LDH) to stimulate glycolytic flux [8, 9, 10, 11, 12]. Similarly, to accommodate the utilization of glutamine, c-myc upregulates the level of expression of sodium-dependent neutral amino acid transporter (SLC1A5), thereby facilitating the transport of glutamate and uptake of glutamine. c-Myc also induces glutaminase GLS which catalyzes the conversion of glutamine to glutamate [8, 9, 10]. We hypothesize that the inhibition of c-myc expression and function, in addition to restriction of access to glucose/glutamine are both bona fide anti-carcinogenic approaches.
\nCancer biology studies, for the past several decades, have focused on the identification and defining of DNA mutations, the narrowly centered focus being underpinned by the viewpoint that cancer is largely a genetic disease. However, cancer cells are also endowed by the acquisition of several hallmarks that enable them to become tumorigenic; among them is the apparent rewiring of gene:metabolite circuitry coordinating an altered metabolic adaptation with dysregulated cell proliferation in cancer cells [13]. Thus, the ability of cancer cells to preferentially utilize glucose and aerobic glycolysis as a paradoxical coincidence with ample oxygen supply was historically made by Warburg in the 1920s [14]. The so-called Warburg phenomenon amply illustrates the oncogenic addiction for glucose as a rapidly established metabolic adaptation that increases the capacity for ATP generation to meet the energy demands for unrestricted tumor cell proliferation and metastasis [7, 13, 15, 16, 17]. A similarly disproportionate reliance on amino acid glutamine is found in various cancer types [3, 4], compared to normal cells [18, 19]. The elevated levels of glutamine metabolism in cancer provide nitrogen for nucleotide and amino acid biosynthesis, and additionally, direct citrate and isocitrate in the TCA cycle for use in lipid synthesis, namely, reprogramming glutamine for energy production and for biosynthetic reactions via anaplerosis [6, 7, 20]. This metabolic rewiring is now recognized as an “Achilles heel” for cancer therapy.
\nc-Myc is a transcription factor that controls the expression of a large number of genes including those involved in ribosome and mitochondria biogenesis, glucose and glutamine metabolism, and lipid biosynthesis [8, 11, 12, 21]. c-Myc controlled gene sets underpin bioenergetics and synthesis of building blocks required for macromolecular assembly, transformation, proliferation and tumorigenesis. As a proto-oncogene, c-myc is frequently amplified/overexpressed in human cancers. Among a myriad of tumorigenic changes, c-myc controls are metabolic reprogramming—preferred use of glucose and glutamine, and sequential regulation of downstream targets/effectors [8, 11, 12, 21, 22]. c-Myc has been shown to induce the expression of glucose transporter Glut1 and glycolytic enzyme LDH to increase glycolytic flux. Similarly, c-myc also activates glutamine transporters SLC1A5 (also known as solute carrier family 1, member 5 and SLC38A5 - solute carrier family 38, member 5) that acts to increase uptake/transport of glutamine [12]. The rewiring of metabolic programming by c-myc provides cancer cells with survival advantage by meeting the demands of energy expenditure, macromolecular syntheses, and catabolic waste disposal. Studies have also shown that upregulation of c-myc is associated with an increase in mitochondrial glutaminolysis, which plays an overarching role on glutamine addiction in cancer cells. c-Myc also induces GLS expression through a dual negative mechanism – suppression of miR-23a/b by c-myc, and inhibition of GLS expression by miR-23a/b [21, 23]. The upregulation of GLS by c-myc facilitates the coordination of metabolic addiction with oncogenesis and the coalescence of metabolic calibration for survival with cellular transformation, proliferation, and cancer-related gene mutations. While metabolic addiction is clinically viewed as favoring carcinogenesis, it is equally plausible that the same nutrient dependence may become a restriction point for designing cancer therapy. Specifically, by limiting access to nutrients within the tumor microenvironments, novel oncogenic targets could become evident and are amenable for rational design of countermeasure strategies. A notable example can be found in the control of c-myc as a “master driver” for cancer; an equally appealing consideration likely involves the downstream targets regulated by c-myc [24, 25].
\nResveratrol is a grape polyphenol whose efficacy has been amply supported by tissue culture and animal studies, and in limited clinical trials [26, 27, 28, 29, 30]. Resveratrol has been reported to inhibit c-myc in multiple cancer types including medulloblastoma cells, breast cancer cells and osteosarcoma [31, 32, 33]. How resveratrol inhibits c-myc via rewiring of metabolite:gene circuitry in tumorigenesis remains largely unknown and will be discussed in the next section. Greater understanding of this will add yet another dimension to the cancer preventive and therapeutic efficacy of resveratrol.
\nSince c-myc is involved in control of metabolic processing and resveratrol can inhibit c-myc expression, we propose that the c-myc-mediated metabolic dysregulation of cancer can be countered using grape-derived resveratrol. It is our hypothesis that resveratrol exerts a dual role in disrupting c-myc-mediated cancer metabolic reprogramming—a direct effect impinging on c-myc expression and stability and associated uptake/transport of glucose and/or glutamine, and an indirect effect involving NQO2 as the mediator affecting c-myc stability via AKT, its downstream effector GSK3β, and by extension control of the activity and function of the proteasome. This provocative assignment for NQO2 greatly expands its cellular role from a cytosolic flavoprotein discovered in 1961 and classically considered phase II enzyme to a multi-tasking regulator involved in cancer cell metabolic reprogramming. Ample data support this postulation. NQO2 requires NRH (N-ribosyl dihydronicotinamide) as the cosubstrate for catalysis; the biosynthetic source of NRH in mammalian cells has not been elucidated suggesting that NQO2 may have other novel cellular functions. Mouse keratinocyte studies show that NQO2 controls TNF-induced NF-kB activation; NQO2 deletion potentiates the induction of apoptosis by abolishing TNF-induced cell survival kinases including JNK, AKT, p38, and p44/p42 MAPK [34]. NQO2 stabilizes C/EBPα degradation mediated by 20S proteasomes [35]. We previously showed that NQO2 is a high affinity target protein of resveratrol: NQO2 binds resveratrol with KD ≤ 50 nM [36]; X-ray crystal analysis shows that binding to resveratrol occurs in a hydrophobic pocket located between dimeric NQO2, possibly where the cosubstrate NRH binds [37]. Since the plasma concentration of resveratrol in humans can reach from 0.5 μM [38] to as high as 4 μM [39, 40], it may be suggested that in vivo levels of resveratrol are sufficient for binding and inhibiting NQO2 enzyme activity and modulation of its other functions. One such novel role may pertain to control of c-myc turnover via T58 phosphorylation by AKT/GSK3β [41, 42, 43], results which agree with/support/resemble our findings using NQO2-knockdown CWR22Rv1 cells, showing that NQO2: (i) inhibits AKT activity and (ii) controls cyclin D1 stability via AKT/GSK3β mediated threonine T-286 phosphorylation [44]. Moreover, oxidized and reduced NQO2 was recently reported to selectively bind DNA-intercalating agents, including ethidium bromide, acridine orange, and doxorubicin; all three agents functioning as inhibitors at nanomolar levels, thereby raising the provocative tenet that NQO2 is a potential regulator of eukaryotic gene transcription and expression [45]. Accordingly, activators/inhibitors of NQO2 may be developed as drug targets for the management of cancers harboring amplified/overexpressed transcriptional factor c-myc. As hypothesized, inhibitors of NQO2 could modify the interplay between NQO2 and c-myc and disrupt the c-myc-mediated growth advantage in cancer cells. If c-myc control is shown to be connected to and under the rubric of genetic (NQO2) and/or chemical (resveratrol) mediated control in glucose/glutamine addiction cancer, then the control of NQO2-c-myc axis by resveratrol may be a promising cancer preventative and therapeutic lead, providing insights on how to better manage and treat glucose/glutamine addicted diseases.
\nAs a powerful factor governing the transcription of large gene sets that encode proteins playing critical roles in numerous cellular processes, both in normal and diseased states, the level of expression of c-myc is under stringent control. Ample data point to c-myc degradation being regulated by sequential phosphorylation of S62 and T58, by two external signal activated kinase cascades, respectively, the RAF/MEK/ERK and PI3K/AKT/GSK3β signaling pathways [46, 47, 48, 49, 50, 51]. T58 phosphorylation of c-myc promotes its interaction with the ubiquitin ligases Fbw7 and Skp2, ubiquitination and degradation by the proteasome [52, 53, 54]. Additionally, deubiquitinating enzymes, USP28 and USP36, also contribute to c-myc degradation [54, 55, 56]. Of note, the reported AKT/GSK3β-mediated c-myc T58 phosphorylation in control of its turnover [41, 42, 43] is relevant to our own studies: (i) NQO2 is involved in AKT/GSK3β-mediated cyclin D1 T286 phosphorylation and degradation and (ii) NQO2 knockdown CWR22Rv1-sh25 cells show a 37% decrease in chymotrypsin-like proteasome activity compared to control CWR22Rv1-sh08 cells [44]. These results suggest a hitherto-never-considered aspect of control of c-myc stability by NQO2. Next, we will discuss a proposed study to test the potential role NQO2 plays as the mediator of control of c-myc stability via AKT/GSK3β-c-myc T-58 phosphorylation, and by regulation of activity and functioning of the proteasome.
\nOur previous studies showing that resveratrol exerts its effects via its target protein NQO2 provide the impetus for testing that down regulation of c-myc by resveratrol requires the participation of NQO2. Based on our data that NQO2 affects cyclin D1 turnover, we expect that NQO2 will increase c-myc degradation, that is, ↓t1/2 instead of conferring protection on c-myc stability by competing against proteasome as has been reported for C/EBPα whose degradation by 20S proteasome is attenuated by NQO2 [35]. Since c-myc degradation by proteasomes is known to involve a multitude of mechanisms, there is a possibility resveratrol or NQO2 may directly affect c-myc degradation by exerting control on ubiquitin ligases like Fbw7 and Skp2 or the deubiquitinating enzyme, USP28. As to whether NQO2 interacts with AKT to affect AKT-GSK3β-mediated c-myc degradation, our expectation is that the accumulated c-myc protein in MG132-treated cells will show a higher T58 phosphorylation as compared to nontreated control cells. As a corollary, addition of GSK3β inhibitor LiCl to treated cells should significantly reduce T58 phosphorylated c-myc protein, in parallel with an increase in c-myc protein accumulation. siRNA-knockdown of GSK-3α or -3β in NQO2 expression cells compared to NQO2 knockdown cells will further confirm the role of GSK3β in mediating c-myc degradation. Results of these studies will provide support for the as yet untested hypothesis regarding the indirect role of NQO2 in controlling AKT → GSK3β → c-myc T58 phosphorylation → c-myc degradation by proteasome, and the direct role of resveratrol acting as a metabolic switch to shut off c-myc-mediated metabolic reprogramming in cancer cells.
\nEpidemiological studies have shown that moderate intake of red wine is correlated with a reduced incidence of dementia and neurodegenerative disease [57]. Moreover, resveratrol, a tri-hydroxyl stilbene found in abundance in red wine, red grapes, peanuts and a number of other consumed foods in the United States, has been reported to confer protection against oxidative stress in PC-12 cells [58, 59]. It has been determined that the preeminent presence of senile plaques, composed mainly of amyloid-β (Aβ) deposits, is a pathological brain feature in individuals diagnosed with Alzheimer’s Disease (AD). The Aβ peptides are derived from cleavage of the amyloid-β precursor protein (APP) and have been shown to destabilize neurons and lead to cell death through the induction of oxidative stress, mediated by the generation of reactive oxygen species (ROS) and elevation in intracellular hydrogen peroxide [60, 61, 62]. Compelling evidence supports that Aβ peptides serve as the “primary instigator” of AD [63]. Davies and coworkers[64] used tissue culture studies combined with biochemical assays and siRNA-silencing approaches to show that resveratrol lowered the levels of secreted and intracellular Aβ-peptides in a concentration and time-dependent manner. Further, this effect occurred not by targeting the Aβ-producing enzymes β and γ-secretases or by affecting the stability of APP or the turnover of its C-terminal fragments; but instead appeared to involve the promotion of intracellular degradation of Aβ via a proteasome subunit β5-dependent mechanism. It is worth noting that resveratrol reportedly also promotes the proteasome-mediated degradation of important regulatory proteins, including cyclin D1 [65], the estrogen receptor-α [66], and the hypoxia-inducible factor-1α [67].
\nThe notion that a folded three-dimensional structure is required for the biological function of a protein is dispelled by the discovery of intrinsically disordered proteins (IDPs) in the 1990s [68]. IDPs can be viewed as proteins that have minimal structures or are devoid of an overall defined fold, either entirely or in parts and are more likely to exist in dynamic, mosaic states under physiological conditions. The absence of structural orderliness also confers plasticity and “fussiness” to IDPs for diverse protein-protein interactions; however, the very same structural flexibility may also render them difficult and challenging as druggable targets using traditional structure-function drug design approaches.
\nEukaryotic transcription factors perform important biological functions in control of gene expression. They play an essential role in identifying the target sequences on DNA located in the vicinity as well as far removed from the transcription start site through direct protein:nucleic acid interaction, and also are required for binding to a large array of co-transcription regulatory proteins via protein:protein interaction. As such, eukaryotic transcription factors have been shown to exhibit a high degree of intrinsic disorderliness; based on bioinformatic model prediction analysis it is estimated that more than 49% of human transcription factors contain intrinsic disorderliness [69]. Studies have also revealed that IDPs, because of their intrinsic destabilized nature circumventing the requirement for unfolding protein substrates for proteolysis by the 26S proteasome, are more likely to be degraded via an ubiquitin-independent mechanism using the 20S proteasomes [70].
\nThe intrinsically unstructured protein (IUP), disorderly theme is also found in transcription factor c-myc; indeed, c-myc only attains an ordered structure after binding to its disordered partner MAX protein (myc-associated factor). Bioinformatics and experimental approaches have estimated that c-myc contains more than 45% of residues which have high probability for disordered structure formation [71]. The possession of intrinsically disordered regions allows c-myc to be degraded independently of ubiquitin, which may account for its observed short half-life at the mRNA [72, 73, 74] and protein levels [75, 76, 77], and is dynamically aligned with its switch on/off master transcriptional role independent of collaborative interaction with the pool of cellular ubiquitin that drives 26S proteasome-mediated protein degradation.
\nIt should be noted that the function of the 20S proteasome can also be modulated by interaction with NAD(P)H:quinone oxidoreductase 1 and 2 (NQO1 and NQO2). Previous studies have reported that NQO1 physically binds the 20S proteasome in an NADH-dependent manner [78, 79, 80] and to protect IDPs from degradation [81]. A double negative feedback mechanism exists between NQO1 and the 20S proteasome [78]. On the one hand, NQO1 acts to attenuate the proteolytic activity of the proteasome; on the other hand, the proteasome degrades the NQO1 FAD-free apo form which manifests as a partially unfolded structure and a substrate for the 20S proteasome [81]. Studies have shown that NQO2 confers protection against proteolytic degradation by the 20S proteasome [35, 82] albeit by a mechanism independent of NQO1 [83].
\nResveratrol has been shown to inhibit the uptake/transport of glucose or glutamine, and decrease the expression of c-myc in cancer cells. Rewiring of metabolite:gene circuitry is a key event in tumorigenesis that has been known for decades [7, 15, 16, 17], however, with an incompletely understood underlying mechanism. In this chapter, we discuss the aversion of c-myc-mediated reprogrammed cancer cell metabolism by targeting the expression and stability of c-myc using a chemical/genetic disruptive approach focusing on resveratrol and its high affinity target NQO2 we identified [36]. Additionally, the hypothesis we propose broadens the classical function of NQO2 in quinone detoxification to AKT/c-myc-mediated metabolic reprogramming observable in a clinical setting. Taken together, resveratrol/NQO2 in a c-myc controlling role to block metabolic addiction represents a novel diet-based chemoprevention approach in concept, and is transformative in implications warranting further investigation. The results will lay foundation for discovery of drugs able to disrupt AKT/c-myc-mediated reorganized metabolism using NQO2 inhibitors.
\nIn summary, we advance the thesis to avert c-myc-mediated metabolic reprogramming in cancer cells by targeting the control of c-myc and the uptake and metabolism of glucose and glutamine and their downstream effectors using resveratrol. We propose to focus on control of phosphorylation of c-myc T58 by GSK3β, shown to be critical for proteasome mediated c-myc degradation, by the resveratrol target protein NQO2 which we have previously shown to act as a modulator of AKT/GSK3β proteasome mediated degradation of cyclin D1 [44].
\nThe dual role resveratrol plays in disrupting c-myc-mediated metabolic reprogramming in cancer cells—a direct role targeting suppression of c-myc expression and an indirect role involving NQO2-AKT-GSK 3β mediated increase in c-myc T58-phosphorylation for increased degradation by proteasome—is illustrated in Figure 1.
\nResveratrol and NQO2 exert dual control of c-myc-mediated glucose/glutamine adaptation in cancer cells by transcription/translation suppression of c-myc expression and by control of proteasome-dependent c-myc stability according to the sequence: NQO2 binds AKT, reducing AKT kinase and increasing GSK 3β activity, resulting in increase in c-myc T58 phosphorylation and facilitating an increase in c-myc degradation by proteasome.
Four hundred years back, Paracelsus stated that, “All substances are poisons; there is none which is not a poison.” If the right dose is taken, it could become a remedy, otherwise poisonous [1, 2]. The therapeutic index or ratio, i.e., LD50/ED50, tells whether the chemical is safe or not.
Poisons are generally found in cases of homicides, suicides, or accidents. They have a significant role to play as the silent weapon to destroy life mysteriously and secretively.
Every poison has almost similar action on the victim’s body. In many cases, they either stop the transfer of O2 to the tissues or create an obstacle in the respiratory system by inhibition of enzymes which are associated with the process. In this, the myoneural junction and the ganglions and synapses are the sites of action. In some cases of insecticidal poisoning, hyperexcitement of voluntary and involuntary muscles can cause death. There are four categories of action of poisons—(i) local action, (ii) remote action, (iii) local and remote actions, and (iv) general action.
Local action: Local action means direct action on the affected site of the body. Examples include irritation and inflammation in strong mineral acids and alkalis, congestion and inflammation by irritants, the effect on motor and sensory nerves, etc.
Remote action: Remote action affects the person due to absorption of that poison into the system of that person. For example, alcohol is absorbed in the system and then it affects the person.
Local and remote actions: Some poisons can affect both local and remote organs. Thus, they not only affect the area with contact to the poison but also cause toxic effect after absorption into the system, for example, oxalic acid.
General action: General action means the absorbed poison affects more than one system of the body, for example, mercury, arsenic, etc.
Toxicity of a poison depends upon its inherent properties such as physiochemical as well as pharmacological properties.
The action of poisons mainly depends upon the following factors discussed below:
Forms of poison: There are three forms of poison:
Physical form: Gaseous/volatile/vaporous forms of poisons act faster than liquid poisons as they are quickly absorbed. Similarly, liquid poisons act faster than solid poisons.
Gaseous or volatile > liquid > solid.
For solid poisons, powdered poisons act quickly than the lumps. For example, there are certain seeds that escape the gastrointestinal tract as they are solid, but when crushed, they can be fatal.
For solids: powdered > lumps
Chemical form: Few substances like mercury or arsenic are not poisonous as they are insoluble and cannot be absorbed when they are in combination with other substances like mercuric chloride, arsenic oxide, etc.
In other cases, the action is vice versa. For example, there are some substances that become inert in combination with silver nitrate and hydrochloric acid and are deadly and poisonous when present in pure forms.
Mechanical combination: The effect of poisons is significantly altered when they are combined with inert substances.
Quantity: Large doses of toxin cause much lethal effect. But this statement is not always true. For example, sometimes when a toxin is taken in very large amount, the body produces a mechanism against it such as vomiting, and thus the intensity of the toxin is reduced.
Concentration: The absorption speed of poison is dependent on concentration; thus poison of higher concentration is fatal. However, there are still some exceptions. For example, a dilute oxalic acid is less corrosive, but the absorption rate is high and so it is more dangerous.
Methods of administration: It has a unique role in the process of absorption. It is fastest through inhalation and then through injection as compared to the oral mode.
Condition of the body: Different persons react differently when exposed to a poison. It is because the condition of our body is also responsible for the increase or decrease of the effect of a poison on the body:
Age: Children and older people are more affected than an adult by the same quantity of toxin.
Sleep: The body functions are slower during sleep; thus toxin circulation in the body is also slower.
Health: Healthy persons can tolerate a toxin better than a weak or ill person.
Dosage: The effect of the poison depends upon its dosage. It is said that the dose determines whether a substance is a poison or remedy. A substance is usually considered a poison after a certain fixed quantity. Although this quantity is not fixed for all people, it is considered according to the average effect on the population. There are two considerable effects of poison on the body of a person; these are the subtle long-term chronic toxicity and immediate fatality.
Some poisons are lethal in microquantities, while others can affect in large doses. The significance of a dose can be understood by taking an example of a metal essential in the food, for example, iron, copper, manganese, zinc, etc.; if its dose is higher than the body requires, it can be lethal.
Effective dose (ED): The effective dose is the quantity of a substance at which it shows its effect in the population. In most cases, ED50 is measured as a dose which induces a response in half of the targeted population.
Lethal dose: The lethal dose (LD) 50 is the amount of drug which is expected to cause death of 50% population.
Hypersensitivity: It is basically the type of reaction initiated by the body against any other substances. Sometimes, it could be related to allergy. There is an assumption that hypersensitivity does not depend on wrong doses. Every person who is hypersensitive to a particular substance has a dose related that defines the quantity required to cause hypersensitivity to that person. The allergic response is actually a toxic response and can be sometimes fatal.
Idiosyncrasy: It is defined as a reaction produced by the body to a chemical genetically. It is a type of person that affects only those people who are genetically sensitized to that particular chemical or substance but will show no effect on others. In such cases, the person experiences discomfort for several hours or if the dose is high can be fatal also. For example- peanut allergy in some people.
Tolerance: It is the capability of a person to not produce any effect against a chemical that usually causes reaction to normal persons. It is a state of reduced or no reaction to a chemical. There are basically two types of mechanism that induces tolerance. First is when the toxin reaches the effective site, its quantity is very less. This is called dispositional tolerance. The second is because the tissues show reduced response to the toxin.
Tolerance can also be achieved if a drug is taken in a small quantity on a regular basis. This can be explained by taking the example of alcohol. When any human consume alcohol for the first time, he/she will show an effect even when the quantity is small, but eventually the effect will decrease and the person can tolerate a large amount also.
Individual susceptibility: It is defined as the different kinds of responses produced by different individuals to a particular harmful compound. It can be due to occupational or environmental factors and exposures. It is determined by complex genetic factors. Its effect depends upon the intensity of exposure. There is a gene uniqueness that varies from person to person; thus the same amount of exposure can show no effect in one individual, cause illness to other individual, and also could be fatal to someone as well.
The route of administration is the path through which a drug, toxin, or poison is taken or administered into the body of a person which is distinguished by the location where any drug is applied. It is mostly classified on the basis of its target:
Topical—which has a local effect
Enteral—which has a wide effect, i.e., affect the whole system
Parental—which follows a systemic action
Poisons are given or taken so that death can occur at once by shock due to stoppage of body’s vital systems. Drug addicts take drugs through inhalation or injection.
Route of administration plays a very important role in determination of death by poison as time in which death occurs are fastest in inhaled poisons, relatively slow in injected and lastly when ingested orally.
Some important features that are considered during the administration of poisons and can make a poison fatal are:
Rate of dissolution of the poison that depends upon the physical form of the poison, i.e., gaseous, vapors, liquid, solid, etc.
The surface area affected at the site of administration of the poison
The circulation rate of blood in that route
The solubility of the poison, i.e., lipid soluble or water soluble
The concentration of the poison
The time required by the poison to be absorbed completely from the site of administration
Routes of administration can be classified into two categories:
Enteral routes/gastrointestinal routes.
Parenteral routes.
Enteral routes: When the drug is administered through the gastrointestinal tract, it is defined as an enteral route. It has both oral and rectal routes. It also includes sublingual and sublabial routes. It is comparatively a slower mode of action for absorption of drugs:
Oral route: Generally absorption takes place in the tongue and the gums of the oral passage. The pH of the buccal cavity and mouth ranges from 4 to 5. Sublingual and supralingual routes have a significant role in absorption. The sublingual absorption is faster as the toxin is transformed directly to the heart, but it takes more time.
Rectal route: Administration of drugs can be done through anus which directly absorbed in bloodstream through membrane of mucous. This administration can cause the burning of tissues or bleeding in rectum as the area is very sensitive.
Parental route: It includes all the other routes that does not involve the gastrointestinal tract. It has a systemic effect on the body. It has the following categories of administration:
Intradermal: Here, the administration of drugs takes place from surface of skin. This type of poisoning is mostly found in chronic poisoning cases.
Intravenous: It is one of the fastest modes of drug administration as the injection is directly taken and the drug is transferred directly into the veins and thus is directly circulated into the blood quickly. Immediate death might be caused by this type of drug.
Intraosseous: It involves an administration of a drug directly into the bone marrow. This mode is actually used for administration of drugs for medical purposes.
Intra-arterial: It involves an administration of a drug into the artery directly through injection. It is a fast mode of administration.
Intramuscular: In this mode, the drug or poison is administered into the muscle of the thigh, upper arm, or buttock. The time required in this mode is greater than other parental modes.
Subcutaneous: In this mode, the drug is injected into the layer beneath the skin, i.e., the subcutaneous layer. The drug then goes to the small blood vessels and then to the bloodstream. This mode is used for mostly those protein drugs that would be destroyed if administered through the gastrointestinal tract.
Inhalation: In this mode, the nose is the primary path. Because of the presence of mucous membrane, the nasal aperture is very absorptive. The microparticles of poisons are easily absorbed and transported quickly to the lungs. From the lungs, they are circulated into the blood.
Poisons are classified into two ways:
Based on their action on the body.
Based on their physical and chemical properties [1].
Classification based upon the effect of poison on the body:
Corrosive: The poisons burn the tissues or organs when they come in contact with them, e.g.:
Strong acids such as H2SO4, HNO3, HCL, etc.
Strong alkalis such as hydroxides of Na, K, NH4, etc.
Irritants: The poisons irritate the tissues or organs when they come in contact with them [3]:
Inorganic:
Nonmetallic phosphorous, chlorine, bromine, iodine, etc.
Metallic salts of arsenic, antimony, mercury, copper, lead, zinc, etc.
Organic:
Vegetable—castor oil, madar, croton oil, etc.
Animals—snake venom, cantharides, insect bites, etc.
Mechanical—glass powder, needles, diamond dust, hair, etc.
Neurotics: Poisons affect the nervous system and the brain [3]:
Cerebral:
Narcotic—opium and its alkaloids
Inebriant (depressant)—alcohol, ether, chloroform, and chloral hydrate
Spinal:
Excitant (stimulants)—nux vomica and strychnine
Depressant—gelsemium
Cardiorespiratory:
Cardiac—aconite, digitalis, oleander, and hydrocyanic acid (HCN)
Asphyxiants—carbon monoxide, carbon dioxide, and hydrogen sulfide
Miscellaneous: A number of chemicals having diverse actions on their body are included in this group [4]:
Animal poisons
Curare (an arrow poison)
Poisonous food articles
Industrial poisons—methyl isocyanate (MIC)
Fuels—petroleum and kerosene
Insecticides—endrin, dichlorodiphenyltrichloroethane (DDT), and
naphthalene
Radioactive substances
Classification of poisons based upon their properties:
Inorganic poisons
Metallic poisons:
Arsenic: It has been the most known and exclusively used throughout
the ages to poison men and animals [1].
It is a white tasteless powder and a pinch of the poisons can kill two adult persons.
Arsenic for homicidal purposes is mixed with various food articles, e.g., cooked food, milk, tea, liquors, or medicines.
Arsenic in a metal form is not poisonous; its oxides are highly poisonous. It is extensively used in insecticides, etc. [5].
Mercury: Chloride and nitrites of mercury are highly poisonous. They
are used in chemical industry and as fungicides.
Lead: Most of its compounds are poisonous. This is a slow poison,
e.g., Sindoor adulterated with red lead oxide.
Copper: Its salts are used in electroplating; copper sulfate is a poison.
Thallium: Thallium salt is used as rat poison [6].
Antimony: Its effect is like that of arsenic.
Nonmetallic poisons:
Cyanides: Cyanides of potassium and sodium are extremely
poisonous, even in small quantities. They react with the acid of
gastric juices in the stomach to form hydrocyanic acid, which
paralyzes the respiratory center in the brain resulting in death due to
respiratory failure [4].
Yellow phosphorus: In olden days it was used in match industry and
several times proved highly poisonous.
Iodine: Only elemental iodine in high quantity is poisonous.
Strong acids and alkalis: These are highly poisonous with corrosive
effects, e.g., sulfuric acid, nitric acid, sodium, potassium
hydroxides, etc.
Gases: Phosphine gas kills rats when used on the rat holes and is
poisonous for infants. MIC killed over 2000 persons and invalidated
several others in a gas leak tragedy in Bhopal in 1984. Some other
poisonous gases are HCN, carbon monoxide, hydrogen sulfide,
arsine, etc. [3].
Organic poisons
Volatile poisons:
Ethyl alcohol: It is poisonous if taken in excess.
Other alcohols: Methyl alcohol and isopropyl alcohol are poisonous.
Methanol, used in polish and chemical industries, is used in illicit
liquor, and its intake causes paralysis, blindness, and death [3].
Phenol: Phenol or carbolic acid could be poisonous. It is mostly used
as a disinfectant [6].
Miscellaneous substances: Various industrial chemicals like
chlorinated hydrocarbons, benzene, chloral hydrate, etc. are
poisonous. In several cases of poisoning, chloral hydrate could be
used in illicit liquors.
Nonvolatile substances:
Alkaloids: Several narcotics and vegetable poisons contain alkaloids,
e.g., strychnine, morphine, cocaine, nicotine, etc.
Barbiturates: These drugs are synthetic and induce sleep [1].
Glycosides: These drugs can cause cardiac arrest and could be fatal
such as aconite, oleander digitalis, etc.
Insecticides and pesticides
Poisoning: It is known as the injurious effect caused by the action of a poison or a detrimental chemical substance. It leads to the development of adverse reaction toward the harmful chemicals or drugs. It is basically differentiated in three categories: suicidal, homicidal, and accidental. Cattle poisoning is the poisoning related to animals. Accidental poisoning is caused by negligence and carelessness. Homicidal poisoning includes the killing of a person due to the poison. Suicidal poisoning refers to the use of toxic chemicals in order to kill oneself.
Corrosive poisoning: It is caused by poisons such as acids and alkalis. They produce a corrosive action on the human body by causing ulcers and acute inflammation.
Metallic poisoning: Metals such as arsenic, mercury, lead, etc., when ingested, cause a deleterious effect. This is known as metallic poisoning.
Plant poison: The study of plant poisons is known as phytotoxicology. Plant poisons, or phytotoxins, comprise a vast range of biologically active chemical substances, such as alkaloids, polypeptides, amines, glycosides, oxalates, resins, toxalbumins, etc.
An alcohol is a drink that contains ethanol. Ethanol is made by fermentation of grains, fruits, and some resources of sugar. Chemically, it is a group of compounds whose saturated carbon chain has a “-OH” group. Alcohol is also a depressant, and in low dose, it can reduce tension, cause euphoria, and improve sociability, but in high dose it can cause stupor, drunkenness, and even death. Regular alcohol intake can cause cancer, alcoholism, dependency, etc. 33% of the total people in the world consumes alcohol. Drinks containing alcohol are broadly classified into three classes, i.e., beer, spirit, and wine, whose alcohol content varies between 3% and 50%. When diluted, alcohol has nearly sweet taste, but when concentrated it gives a burning sensation. 90% of the absorbed alcohol is metabolized by the liver and broken down into less toxic metabolites. Alcohol acts on the central nervous system (CNS) as a depressant on the cells of the cerebral cortex. Its adverse effects like a decrease in cognitive and psychomotive skills are well documented. Alcohol percentage (ABV) differs from one brand to another, for example, beers contain 5%, wines contain typically 13.5%, fortified wines contain 15–22%, spirits contain 30–40%, fruit juice contains less than 0.1%, and cider/wine coolers contain 4–8% ABV [1].
The goal of blood alcohol test is to check the concentration of alcohol in the body. This test result is known as blood alcohol concentration (BAC) which indicates alcohol % in the blood. It is directly proportional to the alcohol in the body, and alcohol hinders with people’s decision, control on them and other characteristics [3]. This test can tell the presence of alcohol in blood for 12 hours [4]. Blood quickly absorbs alcohol and is measured within minutes of consuming alcoholic drink. The highest level of BAC result can be reached within an hour of consuming alcohol. Intake of food can vary the result. Liver breaks down almost 90% of alcohol and rest are given out from exhalation and urine [5].
In case of deaths due to alcoholic intoxication, the viscera is collected and preserved in saturated saline. Preservation of sample is very important as if wrongly preserved it can ruin the examination. Generally, urine and blood are taken as samples.
A sterile needle must be cleaned up by the swab of a nonalcoholic disinfectant like aqueous mercuric chloride and aqueous benzalkonium chloride (Zephiran) before the suspect’s skin is punctured with it. The use of an alcoholic disinfectant either may give false-positive results or may contribute to falsely high alcohol contents of blood. About 5–10 ml of the sample (blood) is taken in a test tube; an anticoagulant such as potassium oxide and EDTA and a preservative such as NaF are added and stored in the refrigerator at 40°C. The anticoagulant will prevent blood from clotting, and the preservative will inhibit the presence of microorganisms. The urine sample is also collected in the usual manner and preserved with 30 mg of phenyl mercuric nitrate for every 10 ml of urine [6].
Ethyl alcohol is isolated from biological materials by acid distillation. Viscera, vomit, stomach contents, and other materials should be analyzed separately. About 50–100 g of the viscera is taken and is finally minced by thin gruel and adding water (3–5 times) and sulfuric acid. It is passed to steam distillation which is generally heating it on the water bath. The condenser and the receiving flask should be well cooled with ice especially in the hot season, the outlet of the condenser being dipped in little water or NaOH solution. Some pieces of pumice stone are stored in the flask to avoid bumping. It is better to collect the distillate in 4–5 fractions, out of which the first one should not exceed 20 ml and the remaining fractions should be 50 ml each. The distillate contains alcohol and other volatile acids, etc. [6].
There are some tests which show the presence of ethyl alcohol in the exhibits.
Also known as triiodomethane reaction, it is used in the detection of CH3CH (OH) which is present in alcohol. There are mainly two types of different mixtures used in this reaction which are mainly chemically equivalent. A pale yellow precipitate occurs if the result is positive [6].
In the above structure, “R” can be hydrogen or alkyl group or any other hydrocarbon group. In case when R denotes hydrogen, then the compound we have the possibility to find is primary alcohol ethanol. Ethanol is the only alcohol that gives an iodoform reaction. In case R is any hydrocarbon group, then it gives secondary alcohol groups. Tertiary alcohol is not able to contain R group because of the absence of hydrogen atom [7].
In 1 ml of distillate, a few drops of 10% NaOH are added dropwise till the solution becomes brown and warmed for a few minutes. A few drops of iodoform solution are added to change the color to yellow. The mixture has to be again heated on low flame/water bath; a yellow-colored precipitate is formed on standing. The precipitate has to be observed under a microscope. Characteristic hexagonal crystals of iodoform are seen which usually shows the presence of ethanol, acetaldehyde, isopropanol which on standing for long time breaks into flower like structure. This test initially involves oxidation followed by substitution and hydrolysis [6].
Add 1 gm of molybdic acid in 25 ml of a concentrated sulfuric acid which has the reagent. Mix 2 ml of this reagent when hot and with 2 ml of distillate. At the junction of both liquids, a ring will be formed which is deep blue in color. On shaking, the whole mixture will become deep blue which is due to ethyl alcohol. This test is very sensitive and it gives a negative result with acetone, acetaldehyde, and dilute solution of methyl alcohol. Only the strong solution of methyl alcohol gives a light blue color after several minutes [6].
Mix two drops of benzoyl chloride with 2 ml of the distillate. Add 10% of sodium hydroxide drop by drop till the solution becomes alkaline. By providing heat the irritating smell of benzoyl chloride will be replaced by sweet fruity odor of ethyl benzoate. Methyl alcohol gives this test also but not the iodoform test [6].
In case of drunkenness, alcohol detection in the body is very important. Observing behavioral abnormalities of the suspect is the best method, but analyzing the breath, blood, and urine is the only way of confirming it. The analysis of breath alcohol can be performed on the spot with the help of breath-analyzer instruments like Alco-Sensor, Breathalyzer, etc. However, the alcohol content of the blood could be determined by using the modified version of the Kozelka and Hine/Cavett method [6].
In recent years, several methods in determining the alcohol in body fluids are described. Kent-Jones and Taylor reported the results of an investigation into the merits of two methods—the micro Cavett and that of Kozelka and Hine. The micro Cavett method is more accurate, but it suffered from serious inconsistencies in reproducibility, but the Kozelka and Hine method is less accurate and more time-consuming but gives good reproducibility.
Nickolls modified the micro Cavett method which appears to give a more accurate result in comparison with the unmodified method. The simplicity of this procedure increases its use for routine work in laboratory [8].
The principle behind this method is the oxidation of alcohol, which is easy with acetic acid in the presence of oxidizing agents such as sulfuric acid and potassium dichromate. Reduction of each mL of N/20 potassium dichromate solution takes place that is equivalent to 0.575 mg of alcohol [6].
This formula is used to estimate the amount in which alcohol is present in the body.
a. For blood analysis
Here, a = Total amount of alcohol absorbed in the body; p = Weight of the person; c = Concentration of alcohol in the blood; r = Constant which is 0.5 in women and 0.68 in men
b. In urine analysis.
Here, a = Total alcohol content present in the body; p = Total weight of the person; q = Alcohol concentration in the urine; r = Constant, namely, 0.68 for men and 0.5 in women [6].
There are several methods in determining ethanol in the blood, urine, and serum. One of the most important methods is gas chromatography (GC). The sample is injected in a heating chamber, and due to its high temperature, alcohol converts in vapors which are carried by inert carrier gas such as nitrogen through the column which is packed by an adsorbent material. Separation of different types of components depends on their different affinity, i.e., partition coefficient toward adsorbent phase which is stationary and later detected as shown in the figure below. A chromatogram so obtained helps in qualitative as well as quantitative analysis [6].
Various components of gas chromatography are [9]:
Carrier gas
Flow regulator
Injector
Column
Stationary phase
Oven
Detectors
Display device
The area covered by the peak represents the amount and position of a particular type of compound [6].
Operating conditions [10]:
Column: Porapak polymer bead 80–100 mesh or its equivalent, which can separate or resolve the ethanol.
Column temperature: 1600°C.
Carrier gas: Nitrogen.
Rate of gas flow: 50 ml/minute.
Detector: Flame ionization detector.
Alternative operating conditions:
Column: 0.3% Carbowax 20 M on 80–100 mesh Carbopak C, 2 m × 2 mm ID or its equivalent.
Column temperature: 350°C for 2 minutes and then programmed at 50°C per minute to 1750°C and hold for at least 8 minutes.
Carrier gas: Nitrogen at 30 ml/minute [6].
The purpose of this chapter is to discuss the mode of action and function of poisons once they reached in the human body. The impacts of poisons are severe and even cause death if not treated properly.
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