Poisons and Drugs

Prof. Monzir S. Abdel-Latif Chemistry Department Islamic University of Gaza http://www.monzir-pal.net

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Essential Substances

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Therapeutic Index

 The

Therapeutic Index

of a drug is the ratio of the toxic to the therapeutic dose. Drugs with a low therapeutic index may only require a small increase in dose to produce toxic effects.

 TI = TD 50 /ED 50 ED 50 is the therapeutically effective dose while TD 50 dose, both in 50% of population is the toxic  The therapeutic index for diazepam is somewhat forgiving, about = 100. Other drugs, however are much less (such as Digoxin, which has an index of 2 or 3 (.

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 Sometimes the term

safety ratio

is used instead, particularly when referring to psychoactive drugs used for non-therapeutic (i.e. nonmedical) purposes.

In such cases, the "effective" dose is that which produces the

desired

effect, which can vary and can be greater or less than the therapeutically effective dose.  ED 50 in this case is the dose that brings the desired effect, while TD 50 has the same definition as before.

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Non-animal Alternative Methods



In vitro

methods and quantitative structure-activity relationship ((QSAR) models for the prediction of acute systemic toxicity have been reviewed in several recent workshops and articles.

 A non-animal replacement method for acute systemic toxicity will have to provide information on many complex biological processes, including toxicokinetics and organ toxicity. 6

Spectrum of Toxic Effects

We have seen earlier that toxic effects can be : 1.

Local or systemic 2.

3.

Reversible or irreversible Immediate or delayed A good example of delayed toxic effects was observed in cases where pregnant women were given diethylstilbesterol (DES) to prevent miscarriages. However, daughters of these mothers developed vaginal cancer after 15-20 years, while males developed prostate cancer and reproductive dysfunction. Therefore, long-term studies are essential not only on subjects taking the drug but on fetus and offspring.

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Toxicity versus Allergic Reactions

Allergic reaction (also known as hypersensitivity and sensitization reaction) to a toxicant results from previous sensitization to that toxicant or a chemically similar one. The chemical acts as a hapten and combines with an endogenous protein to form an antigen, which in turn induces the formation of antibodies. A subsequent exposure to the chemical will result in an antigen – antibody interaction, which provokes the typical manifestations of allergy. Thus, this reaction is different from the usual toxic effects, first because a previous exposure is required, and second because a typical sigmoid dose – response curve is usually not demonstrable with allergic reactions. 8

MOLECULAR TARGETS

Receptors

Receptors are located across plasma membrane, or in cytosol, or nucleus, and serve to transmit physical or chemical signals to the cell. There are many types of receptors serving a variety of functions. Some of them are known to be affected by toxicants.

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Some Receptors

Neurotransmitter receptors

include the cholinergic (nicotinic, located in ganglia and skeletal muscles, and muscarinic in smooth muscles and brain), - and adrenergic, dopamine, opiates, and histamine (H1 and H2) receptors.

Hormone receptors

are for molecules like insulin, cortisone, thyrotropin, estrogen, progesterone, angiotensin, glucagon, prostaglandin, and others.

Certain chemicals such as antidepressants and antitumor agents bind with specific macromolecules that may be considered as “ drug receptors.

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Structure and Signal Transduction

The various functional categories of receptors described above exert their biological effects upon binding with an appropriate ligand, which may be an endogenous or exogenous substance. These effects are preceded by a series of biochemical activities, the signaling, which vary according to the structural characteristics of the receptor. Structurally they may be placed in four classes of receptors. These are: (1) G-protein coupled receptors, (2) ligand-gated ion channels, (3) (4) voltage-gated channels, and intracellular receptors.

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Enzymes

Enzymes are common targets of toxicants. The enzyme effects may be specific, such as the inhibition of acetylcholinesterase (AChE). They may be reversible, such as the case with a number of carbamate insecticides on AChE. Irreversible enzyme inhibition is exemplified by DFP (diisopropyl fluorophosphate), which covalently bind with the enzymes. The effects may be nonspecific. For example, lead and mercury are inhibitors of a great variety of enzymes.

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The last step of the oxidation of many chemicals is catalyzed by the cytochrome oxidase chain. Hydrocyanic acid (HCN) can bind with the iron in these enzymes and block their redox function. The aerobic respiration of cells is then arrested.

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Carriers

Carriers such as hemoglobin can be affected by a toxicant through preferential binding. For example, carbon monoxide can bind hemoglobin at the site where oxygen is normally bound. Because of its greater affinity for hemoglobin, it can inactivate hemoglobin and cause manifestations of oxygen deficiency in tissues. Oxygen transport can also be impaired by an accumulation of methemoglobin, which is an oxidation product of hemoglobin with no oxygen binding ability. In normal individuals, the trace amount of methemoglobin is readily reduced to hemoglobin. Certain toxicants, such as nitrites and aromatic amines, can enhance the formation of methemoglobin. People with glucose-6-phosphate dehydrogenase deficiency have a lower capacity to regenerate hemoglobin from methemoglobin and are thus prone to have methemoglobinemia.

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Structural Proteins

Extracellular structural proteins such as collagen are unlikely to be affected by toxicants. However, toxicants such as ozone and asbestos may cause an increase in fibroblasts and deposition of collagen in the lung. Intracellular structural proteins, such as cytoskeleton (the network of protein filaments and microtubules in the cytoplasm that controls cell shape), may be damaged by toxicants such as arsenic, paraquat, benzene, styrene, and deoxynivalenol.

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Coenzymes

Coenzymes are essential for the normal function of enzymes. Their levels in the body can be diminished by toxicants that inhibit their synthesis. For example, pyrithiamine can inhibit thiamine kinase, which is responsible for the formation of the coenzyme thiamine pyrophosphate. NADPH can be destroyed in the presence of free radicals. Metal-dependent enzymes can be inhibited by chelating agents (e.g., cyanides and dithiocarbamates) through removal of metal coenzymes such as copper and zinc.

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Lipids

Peroxidation

of polyenoic fatty acids has been suggested as a mechanism of the necrotizing action of a number of toxicants, such as carbon tetrachloride, ozone, and estrogen.

The general anesthetics, ether and halothane, as well as many other lipophilic substances can accumulate in the cell membranes and thereby interfere with transport of oxygen and glucose into the cell. The cells of the central nervous system are especially susceptible to a lowering of oxygen and glucose level and are therefore among the first to be deleteriously affected by these substances.

Membrane dissolution can follow contact with organic solvents and amphoteric detergents. The ions of mercury and cadmium can complex with phospholipid bases and expand the surface area of the membrane, thereby altering its function. Lead ion can increase the fragility of erythrocytes and result in hemolysis. The oxygen-carrying function of hemoglobin is lost after it escapes from the hemolyzed erythrocytes.

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Nucleic Acids

Covalent binding

between a toxicant (such as alkylating agents) and replicating DNA and RNA can induce cancer, mutations, and teratogenesis. Such toxicants may also exert immunosuppressive effects.

Antimetabolites (are a major family of cytotoxic substances, that disturb or block one or more of the metabolic pathways

) such as methotrexate may be incorporated into DNA and RNA and then interfere with their replication.

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Others

Hypersensitivity reactions

result from repeated exposure to a particular substance or to its chemically related substances. The latter phenomenon is referred to as cross sensitization. The substance, if it is a large polypeptide, acts as an antigen and stimulates the body to form antibodies. Otherwise, the substance acts as a hapten and combines with proteins in the body to form antigens. The reaction between an antigen from a later exposure and the corresponding antibodies results in the release of histamine, bradykinin, and others. The reaction has a typical pattern irrespective of the nature of the antigen. 19

Corrosive agents

such as strong acids and bases can destroy local tissues by precipitating cellular proteins. Irritation of the underlying tissues occurs as a consequence.

Blockade

of renal and biliary tubules may follow the precipitation of relatively insoluble toxicants or their metabolites. For example, acetylsulfapyridine, a metabolite of sulfapyridine, may block renal tubules, and harmol glucuronide from harmol may produce cholestasis.

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Body weight versus area

On a body weight basis, it is assumed for toxicity data extrapolation that humans are usually about 10 times more sensitive than rodents. On a body surface – area basis, humans usually show about the same sensitivity as test mammals,

i.e.

the same dose per unit of body surface area will give the same given defined effect, in about the same percentage of the population. Knowing the above relationships, it is possible to estimate the exposure to a chemical that humans should be able to tolerate.

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Chemical Interactions

1.

Additive effect The simplest interaction is an additive effect: this is an effect, which is the result of two or more chemicals acting together and is the simple sum of their effects when acting independently. In mathematical terms, 1 + 1 = 2, 1 + 5 = 6,

etc

.

The effects of organochlorine pesticides are usually additive.

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2.

Synergistic (multiplicative) effect

This is an effect of two chemicals acting together, which is greater than the simple sum of their effects when acting alone; it is called synergism. In mathematical terms, 1 + 1 = 4, 1 + 5 = 10,

etc

.

Asbestos fibers and cigarette smoking act together to increase the risk of lung cancer by a factor of 40, taking it well beyond the risk associated with independent exposure to either of these agents.

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3.

Potentiation

In potentiation, a substance that on its own causes no harm makes the effects of another chemical much worse. This may be considered to be a form of synergism. In mathematical terms, 0 + 1 = 5, 0 + 5 = 20,

etc

.

For example, isopropanol, at concentrations that are not harmful to the liver, increases (potentiates) the liver damage caused by a given concentration of carbon tetrachloride.

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4.

Antagonism

The opposite of synergism is antagonism: an antagonistic effect is the result of a chemical counteracting the adverse effect of another; in other words, the situation where exposure to two chemicals together has less effect than the simple sum of their independent effects. Such chemicals are said to show antagonism. In mathematical terms: 1 + 1 = 0, 1 + 5 = 2,

etc

.

For example, histamine lowers arterial pressure, while adrenaline raises arterial pressure 25

Epidemiology and human toxicology

Epidemiology is the analysis of the distribution and determinants of health-related states or events in human populations and the application of this study to the control of health problems. It is the only ethical way to obtain data about the effects of chemicals on human beings and hence to establish beyond doubt that toxicity to humans exists. The following are the main approaches that have been used in epidemiology.

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1. Cohort study

A cohort is a component of the population born during a particular period and identified by the period of birth so that its characteristics (such as causes of death and numbers still living) can be ascertained as it enters successive time and age periods. The term ‘ cohort ’ has broadened to describe any designated group of persons followed or traced over a period of time.

Therefore, the group is selected before the study is done. 27

In a cohort study, one identifies cohorts of people who are, have been, or in the future may be exposed or not exposed, or exposed in different degrees, to a factor or factors hypothesized to influence the probability of occurrence of a given disease or other outcome. An essential feature of the method is the observation of a large population for a sufficient (long) period, to generate reliable incidence or mortality rates in the population subsets. 28

Cohort study

of lung cancer and smoking

Lung cancer cases No lung cancer control Smokers 127 (a) 35 (b) Nonsmokers 73 (c) 165 (d) 400 persons were selected and followed for a long period of time, with regards to smoking and development of lung cancer.

These were classified according to being smoking or nonsmoking, developed cancer or not 29

No. of smokers with lung cancer No. of smokers without cancer Total number of smokers No of non smokers with cancer No of non smokers without cancer Total No. of non smokers Proportion of smokers who develop cancer Proportion of non smokers who develop cancer a b a + b c d c + d a/(a+b) c/(c+d) 127 35 162 73 165 238 0.784

0.307

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Relative risk

 Relative risk (RR) is defined as:  RR = {(a/a+b)}/{c/(c+d)}  RR = {127/(127+35)}/{73/(73+165)}  RR = 2.6

 This means that the probability of developing cancer is 2.6 times as high in smokers as in nonsmokers. This is an evidence of association of cancer with smoking.

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2. Case-control study

A case-control study starts with the identification of persons with the disease (or other outcome variable) of interest, and a suitable control (comparison and reference) group of persons without the disease. The relationship of an attribute to the disease is examined by comparing the diseased and non diseased with regard to how frequently the attribute is present or, if quantitative, the levels of the attribute, in the two groups.

Example: cancer suspected due to nuclear material exposure To examine the effect of exposure of pregnant women to pesticides on the birth weight of the infants 32

Case control study of lung cancer and smoking

Smokers Lung cancer cases 127 (a) No lung cancer control 35 (b) Nonsmokers 73 (c) 165 (d) Total 200 200 200 persons with lung cancer (cases) and 200 persons without lung cancer (control) were selected and categorized to whether they are smokers or nonsmokers 33

Odds (Chance) ratio

      OR is a measure of association between exposure and outcome. OR = (a/c)/(b/d) Odds of exposure among cases = a/c =127/73 = 1.7397

Odds of exposure among control = b/d = 35/165 = 0.2121

Odds ratio = 1.7397/0.2121 = 8.2

This means that the odds of exposure to smoking among cases of lung cancer are 8.2 times as large as the odds of smoking among control. This indicates an important association between lung cancer and smoking. 34

Risk factors

 Risk factors are factors that increase the probability of having the disease  Protective factors are factors that will decrease the probability of having the disease. This is implied by an odd ratio less than 1 35

Case control study of obesity and regularly eating vegetable

Obese cases No obese control Eat vegetable Do not eat veget Total 121 (a) 129 (c) 250 171 (b) 79 (d) 250 36

Odds ratio

     OR = (a/c)/(b/d) Odds of exposure among cases = a/c =121/129 = 0.938

Odds of exposure among control = b/d = 171/79 = 2.1646

Odds ratio = 0.938/2.1646 = 0.43

This means that the odds of exposure to eating veget among obese persons were 0.43 times as large as the odds among non obese control. This indicates that eating vegets could be a protective factor decreasing the probability of obesity 37

Case-control study of depression and regularly eating vegetable

Depressed cases undepressed control Eat vegetable 90 (a) 90 (b) Do not eat veget Total 130 (c) 220 130 (d) 220 38

Odds ratio

      OR = (a/c)/(b/d) Odds of exposure among cases = a/c = 90/130 = 0.6923

Odds of exposure among control = b/d = 90/130 = 0.6923

Odds ratio = 0.6923/6923 = 1.0

This means that the odds of exposure to eating veget among depressed persons were the same as the odds among undepressed control. This indicates that eating vegets has no association with depression.

Conclusion: OR>1 suggests a possible risk factor, an OR<1 suggests a possible protective factor, while an OR=1 suggests no association between exposure and outcome 39

Advantages of a case-control study

 Can be easily used for studying infrequent diseases  Relatively inexpensive as no follow-up is necessary  Fast process, as no need to wait for the accumlation of enough cases as in cohort studies  Cheaper to do than cohort studies 40

3. Confounding studies

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A confounding variable is a variable (say, pollution) that can cause the disease under study (cancer) and is also associated with the exposure of interest (smoking). The existence of confounding variables in smoking studies made it difficult to establish a clear causal link between smoking and cancer unless appropriate methods were used to adjust for the effect of the confounders 42

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An example of a confounding study

  We are studying categories which developed a disease and did not develop a disease as a consequence of exposure. First a table like the one below is constructed: Exposed Not exposed diseased No disease a c b d Total a+b c+d 44

No. of people with disease No. of people without disease Total number of exposed No of unexposed but diseased No of unexposed without disease Total No. of unexposed Proportion of exposed who developed disease Proportion of unexposed who developed disease a b a + b c d c + d a/(a+b) c/(c+d) 45

 Three cases: 1.

a/(a+b) > c/(c+d) This means that exposure and disease are positively associated 2. a/(a+b) < c/(c+d) This means that exposure and disease are negatively associated. Exposure is thus a protective factor 3. a/(a+b) = c/(c+d) This means that there is no association between exposure and disease 46

 The relative risk (RR) is a measure of the degree of association between the exposure and development of the disease  RR = {(a/a+b)}/{c/(c+d)}  RR>1 means that exposed individuals have higher probability of developing the disease  RR<1 means that exposure leads to less risk of the disease, i.e. exposure is a protective factor  RR=1 suggests no association between exposure and disease 47

Bedsore Mortality Example

 9400 cases where identified in a hospital as having or not having bedsores. These were followed to determine whether with regards to death and whether there is an association between bedsores and death.  Among these, 824 have bedsores while 8576 did not have bedsore.

 Among these, 116 were admitted to hospital as high severity cases, while 9284 were low severity cases.

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Detailed info:

     Of the 79 people who had bedsores and died 55 had high medical severity and 24 had low medical severity Of the 745 people who had bedsores and did not die, 51 had high medical severity and 694 had low medical severity Of the 286 people who had no bedsores and died, 5 had high medical severity and 281 had low medical severity Of the 8290 people who had no bedsores and did not die, 5 had high medical severity and 8285 had low medical severity.

Now construct a table for each category: 49

Bedsores association Association of interest Death association Medical severity 50

First: Is medical severity a confounding factor?

 To answer this question we should check whether medical severity is associated with exposure (bedsores) and endpoint (death).

 This can be simply done by constructing the appropriate association tables.

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Association between medical severity and bedsores

Bedsore High Medical Severity Low Medical Severity 106 718 Total 824 No bedsore 10 8566 8576 RR = (106/824)/(10/8576) = 110 Therefore, a strong association is present between medical severity status and bedsores 52

Association between medical severity and death

High Medical Severity die 60 Did not die 56 Total 116 Low Medical Severity 305 8979 9284 RR = (60/116)/(305/9284) = 15.7

Therefore, a strong association is present between medical severity status and death 53

Conclusion

 It is clear that medical severity has strong association with bedsores and death.

Association of interest Bedsores Death RR=110 RR=15.7

Medical severity 54

Now let us look at: Bedsores and mortality

died Did not die Total Bedsore 79 745 824 No bedsore Total 286 365 8290 8576 9035 9400 55

No. of people with bedsore who died No. of smokers with bedsore who did not die Total number of people with bedsore No of people without a bedsore who died No of people without bedsore who did not die Total No. of people without a bedsore a b a + b c d Proportion of people with a bedsore who died Proportion of people without a bedsore who died c + d a/(a+b) (c/(c+d) 79 745 824 286 8290 8576 9.6% 3.3% 56

 RR = (79/824)/(286/8576) = 2.9

 This suggests that the probability of death was 2.9 times as high in people with bedsores as in people without bedsores. This may imply a strong association between bedsores and death.

 Do not rush with conclusions as we have to check the effect of the confounding factor; where when patients where admitted to hospital the medical severity of their cases was recorded, leading to two categories: high severity and low severity groups. 57

 Our task now is to check whether medical severity is a confounding factor or not. Two tables will be necessary, one for each category.

 The point is to study association between exposure (bedsores) and endpoint (death) for each case of medical severity. i.e. one for the high medical severity cases and the other for low medical severity cases. This means that we eliminate the effect of the suspected confounding factor by keeping it constant in both cases.

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High medical severity group

Bedsore died 55 Did not die 51 Total 106 No bedsore Total 5 60 5 56 10 116 59

 The relative risk in the high medical severity group  RR = (55/106)/(5/10) = 1.04

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Low medical severity group

Bedsore died 24 Did not die 694 Total 718 No bedsore Total 281 305 8285 8566 8979 9284 61

 The relative risk in the low medical severity group  RR = (24/718)/(281/8566) = 1.02

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 This process is called stratification where two strata where created. In each stratum, the association between bedsores and death cannot be explained by medical severity because in each stratum medical severity was kept constant.  Stratification is used to adjust for a confounding factor.

 Both relative risks of both strata are close to 1. This means that the risk of death has no association to bedsores, provided that adjustment is made for medical severity. 63

Bedsores association No Association Death association Medical severity 64

   1.

2.

The unadjusted relative risk of 2.9 is thus misleading. Therefore, trying to eliminate bedsores will not affect probability of death. If A does not cause B, then eliminating A will not affect the occurrence of B.

Bedsores are said to be guilty by association.

If the adjusted and unadjusted relative risk have been similar, this means that medical severity did not confound the association between bedsores and death. For confounding to occur: There must be an association between cofounder and the disease There must be an association between cofounder and exposure 65