FOOD TOXICOLOGY

3º Food Science and technology

Gloria Fernández Murillo, Blanca García

Palomino, Ramón Morcillo Martín, Laura
Rabasco Vílchez & Victoria Ruiz Gálvez

TOXICITY OF ETHANOL
 INDEX

INDEX...........................................................................................................................................................2

INTRODUCTION.........................................................................................................................................3

HISTORICAL DEVELOPMENT & USES..................................................................................................3

MECHANISM OF ACTION AND PHARMACOLOGICAL ACTIONS OF ETHANOL..........................4

PHARMACOKINETICS...........................................................................................................................4

PHARMACODYNAMICS.......................................................................................................................5

ETHANOL TOXICITY.................................................................................................................................6

PERCENTAGE OF ALCOHOL EFFECT................................................................................................6

ACUTE TOXICITY..................................................................................................................................6

CHRONIC TOXICITY..............................................................................................................................7

EVIDENCE FOR ETHANOL-RELATED OXIDATIVE STRESS.......................................................11

SOURCE OF REACTIVE OXYGEN SPECIES GENERATED BY ETHANOL.................................12

STUDIES WITH ANIMALS......................................................................................................................14

STUDIES WITH RATS..........................................................................................................................14

STUDIES WITH RABBITS....................................................................................................................16

PHARMACOLOGICAL INTERACTIONS...............................................................................................17

DISADVANTAGES & BENEFITS............................................................................................................18

Bibliography................................................................................................................................................20

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 INTRODUCTION

Ethanol, also known as ethyl alcohol, is a chemical compound that has different uses. We can find ethanol
in alcoholic beverages, medicines, cosmetics, fuels, chemical industries...

Next, we will talk about the most general aspects of ethanol, including toxicity, mechanisms of action,
benefits, etc.

HISTORICAL DEVELOPMENT & USES

Ethanol or C2H6O are a colorless, volatile liquid with a characteristic odor and spicy taste. Its
composition: C: 52.24 %; H: 13.13 % y O: 34.73 % and the molecular weight is 46.07 g/mol.

But, all of the above are general characteristics that we already knew. We will now focus on the origin of
ethanol.

The fermentation of sugar into ethanol is one of the earliest organic reactions that man learned to carry
out.

Dried ethanol residue have been found on 9000 year old in China which indicates that Neolithic people in
this part of the world may have consumed alcoholic beverages.

Beer and wine will normally not develop an alcohol content over 15% alcohol by volume, since a higher
concentration makes it impossible for most yeasts to reproduce. Eventually, humans found out that a
higher ethanol concentration could be obtained through distillation.

Distillation is a process where a mixture is separated into various components based on their individual
volatility. Fermented solutions have been distilled since ancient times in order to produce distilled
beverages with a high ethanol content.

The Greeks did not call distillation by this name, but by the Greek word that means to boil. In Egypt and
Persia, distillation techniques became increasingly specialized and complex.

At the beginning of the thirteenth century, the Church already allowed alcoholic beverages to be
manufactured in monasteries using distillation. These drinks (generically called water of life) consisted of
a mixture of water, ethyl alcohol, and aromatic herbs that were cultivated in the vicinity of the
monasteries.

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The year 1796 is significant for ethanol history because this is when obtained pure ethanol by filtering
distilled ethanol through activated charcoal. Ascertain that ethanol consists of hydrogen, oxygen and
carbon, but it wasn't until the early 19th century that the chemical formula was determined. Ethanol in
alcoholic beverages has evolved with history to reach what we know today.

Ethanol can be synthesized in other ways, even though we have focused only on its origin as a result of
alcoholic fermentation.

Ethyl alcohol can be obtained in two ways, such as:

 Most of the world's production is obtained from the processing of biological material, in particular
certain plants with sugars. The ethanol thus produced is known as bioethanol.
 On the other hand, ethanol can also be obtained by chemical modification of ethylene.

This chemical compound can be found in different sites and products. Some examples:

 Alcoholics averages. About this product have been talking before.

 Fuels. More than 90% of the gasoline used in the United States contains ethanol.
 Food additives. It can help to uniformly distribute food coloring, as well as enhance the flavor of
food extracts.
 Home's products. Ethanol mixes easily with water and many organic compounds, and generates an
effective solvent
 Personal care products. As for example cosmetics, toothpaste...
 Medicines.

MECHANISM OF ACTION AND PHARMACOLOGICAL ACTIONS OF

ETHANOL

PHARMACOKINETICS

After consuming alcohol, absorption takes place mainly in the small intestine, but the emptying of the
stomach and the rate of intestinal absorption depend on several factors. The rate of absorption is
accelerated proportionally to the increase in the alcohol concentration ingested, up to a maximum of 40%,
from which a delay in gastric emptying appears with the consequent slowing of absorption, an effect that
also appears in the ingestion of alcohol accompanied by food.

Once absorbed, it is distributed throughout the body. The maximum concentration is in lipid-rich tissues.
Ethanol undergoes a hepatic metabolism and only 2% is eliminated without bio transforming in the urine

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and in the lungs. Pulmonary excretion of ethanol as a mechanism of elimination is of little interest. But
from the analytical and judicial point of view is of great importance, because the methods of bloodless
analysis are based on the determination of ethanol present in the exhaled air. It has been calculated that
the ethanol present in 2000 ml of expired air is equivalent to that in 1 ml of arterial blood.

Ethanol is metabolized by non-microsomal oxidation in the cytosol and by microsomal route. The enzyme
alcohol dehydrogenase transforms ethanol into acetaldehyde and is inhibited by 4-methylpyrazole or
fomepizole. The enzyme acetaldehyde dehydrogenase transforms acetaldehyde into acetic acid which, in
the form of acetyl CoA, enters the Krebs cycle, serves for the formation of ketone bodies, synthesis of
cholesterol and derivatives, and synthesis of fatty acids and drug conjugation. Acetaldehyde
dehydrogenase is inhibited by disulfiram, diethyldithiocarbamate, citrated calcium carbimide,
metronidazole, nitrofurans, sulfonylureas and some cephalosporins. The plasma disappearance kinetics of
ethanol is 8 to 12 ml per hour.

The blood alcohol concentrations achieved in usual situations of social life, range between 50 and 75 mg
per 100 ml of blood. With this concentration the subjective state is usually of pleasant tranquility and a
slight degree of sedation. Binge signs usually appear at concentrations of 100 to 200 mg per 100 ml and
intense intoxications are observed with levels higher than 200 mg per 100 ml; with concentrations of 400
mg per 100 milliliters stupor, coma or both occur; and concentrations greater than 500 mg per 100 ml. of
blood usually cause death.

PHARMACODYNAMICS

Ethanol is a non-selective depressant of the Central Nervous System. As it is a substance much more
water soluble than inhalational anaesthetics, the period of induction of narcosis is prolonged a lot and the
surgical period itself practically coincides with the respiratory paralysis phase, therefore ethanol has not
been used as anaesthetic drug.

Ethanol is the membrane stabilizing drug, dissolving into a lipoidal component, inhibits the active
transport of sodium, potassium, amino acids, catecholamines, etc. It also decreases ATP-ase activity of
sodium-potassium dependent membrane and decreases the use of ATP and oxygen consumption. Like
other non-selective Central Nervous System depressants, ethanol potentiates the inhibitory effects of
gamma aminobutyric acid (GABA) in the Central Nervous System and inhibits the NMDA receptor of
aspartate glutamate and therefore reduces Glutamatergic activity.

Regarding the pharmacological actions, ethanol produces a phenomenon of euphoria, elevation of the
vital tone, superficial improvement of the association of ideas, decrease of self-control and self-criticism,
reduction of vision, muscular incoordination and alterations of the reflexes, decrease in sensation of

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fatigue, decrease in convulsive threshold. As for the cardiovascular system, ethanol is a vasodilator drug
for promoting the release of histamine, although this effect occurs with high doses. The decrease in
angina pain is due more to its euphoric and analgesic effect than to its coronary vasodilator effect.
Ethanol produces cutaneous vasodilatation, which may favour thermolysis and decrease in body
temperature. At low or moderate doses, ethyl alcohol is an antiplatelet agent, reduces the plasma
concentration of low density lipoproteins (LDL) and increases the plasmatic concentration of high density
lipoproteins (HDL).

Ethanol stimulates the hydrochloric secretion, although at high concentrations for a long time it produces
atrophy of the secretory cells. In the liver, ethanol increases the NADH / NAD ratio by inhibiting
glycogenesis with hypoglycaemia and steatosis. Ethanol inhibits the release of antidiuretic hormone
(ADH) and oxytocin by acting on the hypothalamus, ethyl alcohol increases the release of beta-boron
acting on the anterior pituitary and at high doses increases the secretion of catecholamines and hormones
of the adrenal cortex.

On sexuality, ethanol increases libido, but sexual potency decreases. On the other hand, it is uterine
spasmolytic because it inhibits the hypothalamic release of oxytocin. Ethanol interferes with the secretion
of luteinizing hormone (LH) by reducing the serum testosterone concentration. It inhibits the
hydroxylation of testosterone in the testes because NADH accumulates in them by the activity of alcohol
dehydrogenase.

ETHANOL TOXICITY

PERCENTAGE OF ALCOHOL EFFECT

 20 - 50 mg% Decreases fine motor control

 50 - 100 mg% Alter judgment
 100 - 150 mg% Difficulty walking and balance
 150 - 250 mg% Lethargy and difficulty sitting without help
 More than 300 mg% Eat in novice drinkers
 More than 400 mg% Respiratory depression

ACUTE TOXICITY

In the central nervous system depression of frontal cortical areas in system central nervous system
explains euphoria, mild incoordination and abnormal functions such as judgment, reasoning and
behaviour (disinhibition), so they affect the attention, concentration, memory and occasionally
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uninhibited behaviours and antisocial. If the intoxication progresses visual and motor alterations will
appear, inducing respiratory depression and affecting the consciousness state to produce coma (if they
ingest sharply large amounts).

At the musculoskeletal level, rhabdomyolysis can be found as a result of a prolonged immobility.

In the cardiovascular system, arrhythmias may also be present, there are also signs such as facial flushing,
tachycardia and hypotension.

Gastrointestinal disorders: increases the motility of the small intestine and decreases the absorption of
water and electrolytes. By direct irritative effect it causes gastritis, esophagitis, ulcers, digestive tract
haemorrhage, vomiting and can occur a Mallory Weis syndrome.

Acute pancreatitis is a frequent complication in patients with a history of alcohol abuse. The mechanism
that explains this injury is given by an increased secretion of pancreatic proenzymes, increased lysosomal
activity and a decrease in the inactivation capacity of trypsin inside the gland with impaired excretory
function of the Golgi apparatus. These situations activate the enzymes and coagulation factors and
mediators of inflammation that trigger the attack of pancreatitis.

Pulmonary complications secondary to bronchoaspiration. Hypothermia, diaphoresis, mydriasis,

dysarthria, emotional lability, nystagmus.

Seizures may occur, especially in children, due to hypoglycaemia caused by ethanol.

Other metabolic disorders after acute intake of ethanol include acidosis metabolic, hypokalemia,
hypomagnesemia, hypocalcemia and hyperamylasemia.

Nutritional alterations: ethanol lacks nutritional value 1gr of ethanol possesses 7.1 kcal. The fasting
ingestion of ethanol in healthy patients produces transient hypoglycaemia from six to 36 hours by
inhibition of gluconeogenesis.

The acute attack of gout is presented by stimulation of lipogenesis, which triggers an increase in lactate
and fatty acids. To increase the lactate / pyruvate ratio hyperlactoacidemia occurs, which leads to
decreased renal excretion of uric acid which generates hyperuricemia and the production of drop attack.

Metabolic ketoacidosis is a complication due to excessive production of ketones, by increasing the liver's
energy production from the fatty acids generating a large amount of ketoacids such as beta-
hydroxybutyric and acetoacetate. Each step from ethanol to acetaldehyde and acetic acid generates the
production of NADH. The use of NADH decreases NAD and also increases lactate, basic mechanism of
alcoholic ketoacidosis.

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 CHRONIC TOXICITY

At the level of the Central Nervous System conditions:

The Wernicke-Korsakoff Syndrome: Related to vitamin B1 deficiency, caused by gastrointestinal

alterations symptoms of the chronic alcoholism; is a clinical complex of biochemical and structural
alterations conformed by Wernicke encephalopathy and psychosis of Korsakoff.

 Wernicke encephalopathy is of an earlier presentation and starts with faults in the orientation,
apathy and indifference, diminution of reflexes until areflexia. Clinically, it is characterized by
the presentation of a symptomatic triad composed of oculomotor alterations, ataxia and
confusion. The most frequent oculomotor faults they are horizontal nystagmus, paralysis of the
external rectus and paralysis of conjugated gaze.
The lesions of this encephalopathy are of symmetric and periaxial distribution and they locate
in the periventricular zones of thalamus and hypothalamus, mamillary bodies, periaqueductal
white matter and cerebellar vermis. This first phase is reversible with the correction of the
vitamin deficit.
 Korsakoff's psychosis is the second phase of this syndromatic complex that compromises the
antegrade memory that alters learning and retrograde memory recent so that events of previous
months and years are forgotten; the memory immediate is conserved. Clinically it is
characterized by antegrade and retrograde amnesia, distortion of time, confabulation since the
patient can`t structure their memories, apathy and impossibility of integrating into society.

Morel laminar cortical sclerosis: This pathology clinically characterized by a cerebral

pseudoparalysis and a psychotic picture, is related to the previous presence of a chronic alcoholism
picture. Neuropathologically it is characterized by loss neuronal and gliosis in the cerebral cortex.

The Marchiafava-Bignami disease: Although its etiology is unknown, it manifests almost

exclusively in alcoholic patients, undernourished and especially in the chronic consumption of red wine.
It was classified by demyelination and posterior necrosis of the corpus callosum and subcortical gray
matter.

It can occur in acute or chronic alcoholism. It is manifested clinically by dementia, spasticity, dysarthria
and unstable gait.

The alcoholic cerebellar degeneration: It is the pathology in its anatomopathological presentation

is similar to Wernicke encephalopathy. It is the alteration of the upper antero vermouth with neuronal
loss, glios and the reduction of the dendritic branches of Purkinge cells. Clinic presented ataxia with
increased support base, instability and incoordination the lower limbs.
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 Alcoholic dementia: This pathology is related to the abusive and chronic consumption of ethanol.
Initially an established dementia function is presented. Anatomopathologically evidence of atrophy of the
frontal lobes. The relationship with its mechanism of toxicity, the ethical effect, the control of ethanol on
the neuronal membranes, the dehydration of the neurons by ethanol effect, cranial microtrauma, repetition
and nutritional deficiencies typical of chronic alcohol.

Stroke is an alteration in the contraction of blood vessels, ethanol, vasoconstrictor, metabolite, action and
vasodilator action.

Alterations in the peripheral nervous system:

Among the main alterations that have been described are: the alcoholic peripheral polyneuropathy: it is
the most frequent sequel of the chronic alcoholic disease; caused by the deficiency of thiamine and other
B vitamins and by the direct effects of alcohol and acetaldehyde on the neuronal membrane. Its main
lesion is the axonal degeneration of the myelinated and unmyelinated fibers, which clinically translates
into distal, symmetric and mixed neuropathy that mainly affects the lower limbs producing loss of muscle
strength, burning sensation in plants and painful paresthesias. There is alteration of the sensation: it is
tactile, discriminatory and vibratory.

The syndrome of alcoholic dysautonomy:

Related to the direct effect of ethanol and acetaldehyde on the neuronal membrane. Clinically it presents a
combination of cardiovascular, digestive, genitourinary and endocrine symptoms. Among the clinical
findings are sustained tachycardia, extrasystoles, orthostatic hypotension, diarrhea, impotence, urinary
incontinence and disorders of sweating.

In the Gastrointestinal System, various alterations occur:

Alcoholic fatty liver: Occurs in frequent drinkers. It is reversible upon cessation of consumption.
It is a fat accumulation produced by the loss of efficiency in the oxidation of fatty acids and the increase
of their use to esterify them to triglycerides, associated with a decrease in the synthesis and secretion of
lipoproteins. Clinical manifestations are minimal or absent.

Alcoholic hepatitis: Is the precursor of cirrhosis. It is caused by an inflammatory lesion

characterized by hepatic infiltration with leukocytes, liver damage, hepatocyte necrosis and alcoholic
hyalinization. The sequelae of fibrosis are irreversible. Its clinical manifestations can be mild to severe,
including: anorexia, nausea, vomiting, weight loss, abdominal pain, jaundice, fever, cutaneous arterial
angiomas, ascites, edema, encephalopathy, and hemorragia de vías digestivas.

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 Alcoholic cirrhosis: Is an advanced stage that occurs in liver disease by chronic consumption of
ethyl alcohol. Hepatocyte destruction occurs with a connective tissue formation that replaces them
(nodule formation). It is manifested by portal hypertension, esophageal varices, decreased liver size and
induration, generating vascular fibrosis, anorexia, malnutrition, decreased muscle mass, fatigue,
weakness, intercurrent infections, chronic jaundice, hepatic coma, acute and chronic renal failure.

Chronic Pancreatitis: Clinically characterized by persistent abdominal pain, exocrine insufficiency

and diabetes. It usually presents after recurrent acute pancreatitis. Microscopically, pancreatic fibrosis and
irregular calcification occur with destruction of the exocrine parenchyma and endocrine.

Malabsorption Syndrome: Ethanol in chronic consumption alters the absorption of minerals,

vitamins and other nutrients mainly in the small intestine. It decreases the blood concentration of
potassium, magnesium, zinc, phosphorus and calcium. These deficiencies give rise to multiple clinical
alterations.

At the level of the Cardiovascular System, multiple alterations related to the chronic consumption of
ethanol have been reported, among which are cardiomyopathies, arrhythmias and congestive heart failure.

In chronic alcoholics, cardiac disorders such as extrasystoles and other arrhythmias evidenced in the
electrocardiogram have been reported, which seem to be related to vitamin B1 deficiency, interference of
ethanol on calcium channels, the action of acetaldehyde on mitochondria and interference in the synthesis
of the ATP. The previous physiopathological mechanisms are manifested in the muscle fibers, where
fragmentation of fibrils, hyaline and granular degeneration originates.

At the level of the Musculoskeletal System:

Alcoholic myopathy is characterized by muscular atrophy of more than six months of evolution,
especially in the muscles of the shoulder girdle and the pelvic girdle. Clinically, it is characterized by pain
and edema in the muscles, with increased CPK and myoglobinemia. The anatomopathological analysis
shows myonecrosis, presence of lipid vacuoles in type 1 fibers, inflammatory infiltrate, and later
disappearance of lipid vacuoles and appearance of atrophy of muscle fibers type II B. In men, prolonged
consumption of ethanol can cause osteoporosis, related to vitamin D deficiency and calcium, also the
deficiency of magnesium causes alterations in the secretion of the paratyphoid hormone.

At the level of the Endocrine System, calcium alterations (fractures and osteopenia), increased cortisol,
inhibition of vasopressin secretion, decreased T3 and T4, and hypoglycemia have been described.

In the Reproductive System in man, testicular atrophy, gynecomastia, impotence, sterility and
testosterone decrease may occur. In women amenorrhea, ovarian atrophy, absence of corpus luteum and
sterility.
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In the Renal System a diuretic action has been demonstrated by inhibition of tubular reabsorption and
reduction of antidiuretic hormone at the ventricular supra-optic level.

In the Hematopoietic System produces toxic agranulocytosis that leads to anemia, leukopenia and
thrombocytopenia, decreasing platelet aggregation and inhibition of Thromboxane A2. Decreases the
delayed hypersensitivity response. In chronic alcoholics, cellular immunity is affected, presenting
absolute lymphopenia that mainly affects T lymphocytes, helper and suppressor cells and "natural killer"
cells.

It is worth mentioning the effect Mallamby: produced by an acute intolerance to ethanol during
ingestion, where the levels of ethanol in blood are progressively higher. The chronic alcoholic patient
remains sober with abnormal alcohol and fatal. This effect is produced by prolonged, frequent and
excessive consumption.

Pellagra: has been related to nicotinic acid deficiency in chronic malnourished serious alcoholics
and in tuberculous patients treated with Isoniazide which is necessary for the synthesis of NADH and
NADPH; this pathology is characterized by triad dermatitis, diarrhea and dementia. Pellagra has also been
described in alcoholic patients associated with Wernicke's encephalopathy and Marchiafava's disease.
Bignami. (CENTRO DE INFORMACION TOXICOLOGICA DE VERACRUZ)

EVIDENCE FOR ETHANOL-RELATED OXIDATIVE STRESS

Levels of glutathione and related enzymes:

Dramatic reductions in hepatic glutathione levels following acute or extended exposure to ethanol have
been reported for liver (Videla LA, 1982). Glutathione peroxidase is also depressed in the liver following
chronic ethanol dosing (Schisler NJ1, 1989). Lesser reductions, and also increases, in glutathione levels
have been found in brain (Guerri C, 1980). Since compensator processes can be rapid after induction of
excessive ROS, both reductions and increases in cerebral glutathione are regarded as indices of oxidative
stress (Adams, 1989). The turnover of glutathione is elevated after chronic ethanol treatment.

Lipid peroxidation and evidence of damage to other macromolecules:

Lipid peroxidation is an indirect measure which reflects the consequences of ROS activity upon lipid-rich
cell membranes. Results are often reported in terms of thiobarbituric acid reactive materials such as
malondialdehyde. Elevations in lipid peroxidation following ethanol treatment have been frequently
reported for both brain and liver (NR., 1973). Acetaldehyde treatment also elevates hepatic lipid
peroxidation (MU, 1985). Evidence of widespread systemic elevations in lipid peroxidation have also
been detected in vivo by assay of exhalation of metabolically produced ethane (Müller A, 1983). The
attenuation of some of these ethanol-induced changes by the lipid-soluble antioxidant vitamins A or E,
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and the depletion of antioxidant vitamins in ethanol treated rats are consistent with the presence of
induced oxidative stress. The metabolism of ethanol has been shown to lead to the formation of oxidative
products capable of cleaving DNA (Rajasinghe H1, 1990). The capacity of free radicals to facilitate
expression of proto-oncogenes (Maki, 1992) may underlie this finding. This may account for the weakly
carcinogenic properties of ethanol, and also its teratogenic actions. (Blakley PM, 1984). However, there is
no evidence for ethanol causing DNA damage in intact animals.

Iron mobilization:

Iron is well-recognized as an initiator of intracellular ROS production. This may be by way of catalyzing
the generation of the hydroxyl radical from peroxides by the Haber-Weiss reaction, or by direct formation
of the perferryl radical. Free ionic, or incompletely sequestered iron may be essential for the appearance
of ethanol-induced ROS. Several investigators have found ethanol has an effect in the liberation of low
molecular weight iron from bound intracellular reserves.

SOURCE OF REACTIVE OXYGEN SPECIES GENERATED BY ETHANOL

Also, ethanol involves the induction of plenty enzymes related to oxidant status, specifically in liver and
brain. The catabolic steps involved in the degradation of ethanol are candidates for the origin of excess
ROS. Enzymes with ROS-producing potential are shown in Figure 1. Two major classes of enzyme need
to be considered; those oxidizing ethanol to acetaldehyde, and those completing the oxidation to acetate.
(Bondy, 1992)

Alcohol dehydrogenase:

This relatively non-specific soluble

enzyme is considered the primary
initial step in the catabolism of ethanol.
There is evidence that the activity of
several dehydrogenases, including
alcohol dehydrogenase, can bring about
ROS formation, despite the fact that
oxygen is not directly involved (Mattia
CJ, 1993). The mechanism underlying
this may involve the formation of
hydroperoxyl radical by the zinc moiety found in this enzyme. The inhibition of ethanol-effected free
radical production by an inhibitor of alcohol dehydrogenase, 4-methylpyrazole, suggests that ROS are
generated by this enzyme. (Bondy, 1992)

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Catalase:

The evidence that this soluble enzyme is involved in ethanol degradation is that pre-treatment of rats with
ethanol can block the inhibition of cerebral catalase normally brought about by 3-amino-1,2,4-triazole.
This inhibitor can also attenuate some behavioural effects of ethanol, further suggesting the relevance of
this route of oxidation to the brain. (Bondy, 1992)

Mixed function oxidases:

After chronic consumption of ethanol, or in its presence at a high concentration, its metabolism by hepatic
microsomal P-450 enzymes becomes more pronounced. Several of these enzyme species are able to
oxidize ethanol but cytochrome P-450 11E1 (P-450j) is the most selective. This enzyme is inducible by
ethanol and can generate oxidizing species in the absence of substrates as long as NADPH is present.
Under such conditions, cytochrome P-450 11E1 has been reported to exhibit an unusually high rate of
oxidase, and hydrogen peroxide generating activity. (Bondy, 1992)

Another potential route of ethanol oxidation involves the elevation of NADH levels that can occur after
alcohol consumption. The consequent inhibition of fatty acid oxidation allows increased triglyceride
formation and fat deposition in the liver, which may predispose this tissue to peroxidative events.

Aldehyde dehydrogenase:

This mitochondrial NAD+ dependent enzyme constitutes the major means of oxidation of acetaldehyde
by the liver. It is also present in brain. This dehydrogenase is capable of producing hydroxyl ions.
Inhibition of this enzyme by sodium cyanamide, prevents the onset of ethanol-stimulated hepatic lipid
peroxidation. (Bondy, 1992)

Aldehyde oxidase and xanthine oxidase:

Both xanthine oxidase and aldehyde oxidase are molybdenum- and flavin-containing enzymes. The
presence of the latter enzyme in the brain is equivocal but xanthine oxidase, which can also oxidize
acetaldehyde, is present in all tissues. This enzyme is derived by the proteolytic and oxidative
modification of xanthine dehydrogenase. Ethanol treatment may also bring about this enzyme conversion,
and acetaldehyde appears to be the agent directly responsible for this effect. The affinity of aldehyde
oxidase toward acetaldehyde, is much greater than that of xanthine oxidase. By use of various selective
inhibitors of aldehyde oxidase and xanthine oxidase, it was concluded that, in the liver of the ethanol-
treated animal, both enzymes were roughly equal in their ability to promote free radical generation.
(Bondy, 1992)

Ethanol-stimulated lipid mobilization:

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Ethanol has been shown to activate phospholipases Al and A2, in an isolated cardiac preparation. This
effect is blocked in the presence of α-tocopherol. The liberation of arachidonic acid by phospholipase A2
sets in motion a range of oxidative catabolic processes; the ‘arachidonic acid cascade’. This
polyunsaturated fatty acid contains four ethylenic bonds and is readily oxidable. The enzymic conversion
of this compound to prostaglandins, leukotrienes and thromboxanes by cyclooxygenases and
lipoxygenases leads to considerable ROS generation. (Bondy, 1992)

To conclude we could say that ethanol enhances Ros production and as a consequence metabolic stress
leading to several diseases.

STUDIES WITH ANIMALS

To study the toxicity of ethanol, experimental toxicology studies are carried out in vivo using animals. In
this case we are going to comment firstly de different studies that are developed in rats to know their
behaviour when they consume alcohol and secondly, it is going to be comment a study that has been done
in rabbits.

STUDIES WITH RATS

The investigations with rats and mice are a very important tool if we take into account that both humans
and rats come from a common mammal ancestor.

Talking about investigations using rats and mice to study the alcoholism, they can be classified in two
different groups according to the control that the animal has over its consumption: “auto-administration
models” and “forced administration model”.

Auto-administration models:

There are two ways to obtain the auto-administration of alcohol in animals: “box-home models” and
“operating restriction”. Both models have in common that the animal can control the ingestion of alcohol,
the dose and the temporary patrons of the ingestion. However, they are different according to the
behaviour required to obtain the alcohol and the administration route. These models are useful to analyse
the human behaviour because drinkers usually consume alcohol when they can control de amount as well
as the consumption patrons.

As rodents don’t consume alcohol spontaneously, it’s necessary use a procedure to help them to start to
include the drug to their diet, to stabilize its consumption and develop it pharmacological effects.

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 Box-home models: These models allow the animal to access to the drug during 24h or during
limited periods of time where they live, getting close to the drinking trough. This method has been used to
achieve the voluntary consumption and to stablish the phases of the alcohol consumption in three
different stages:
Acquisition phase. During this phase the consumption of the drug occurs during the first two
weeks of exposition. In this stage, the consumption is controlled and the ingestion is exploratory. Talking
about the patrons of consumption and the daily dose, both are unstable and unpredictable. Maybe, during
this phase, animals learn to evaluate the psychotropic effects of the alcohol and adjust their consumption
behaviour.

Controlled consumption phase. When animals have free continuous access to the alcohol, each
one of them develop an individual stable patron of consumption (Wolffgramm, 1990). This patron of
consumption is determinated because the interaction of external factors or the inner state of the animal
(high anxiety levels, genetic…) and would be related with the psychotropic effects of the alcohol
(Spanagel, 2000). Finally, in this stage the effect of the exposition to diverse stress factors are studied to
evaluate the changes produced in the addictive answer.

Loss of control or point of “no return”. The addictive behaviour is defined as the loss of control
and not as a high alcohol consumption. In this step, both external and inner factors don’t influence in the
alcohol ingest. The transition from the controlled consumption to the not controlled seems to be
irreversible in rats, which is a point of no return.

Operating restriction models: These models are used to study the behaviour related with the “research” of
alcohol, in which the access to the drug depends on the behaviour of the animal under the requirement of
an instrumental answer. The advantage of these models is that the investigator can vary the complexity
and the effort that the animal has to carry out to obtain the alcohol by different reinforcement programs
by pressing a lever.

Reinstatement model. It is used to study the process related with the compulsion and relapse to the
drug consumption. In a first step, animals are trained to press a lever to obtain alcohol. After that, there is
an extinction phase, in which pressing the lever is not related with the obtaining of alcohol. In this step,
the experimenter develops different incentives and evaluates which one of them reinstall the answer of
pressing the lever although the drug is not present. There are at least three types of incentives that
produce the reinstall: the administration of a small quantity of alcohol, the stress and conditioned
incentives that were associated with the drug during the first phase. It is considered that pressing again the
lever in the last step is a similar behaviour to the research of the drug and relapse in the human
consumption (Spanagel, 2003)

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 Cut point. This model has been developed to stablish independent measures for two different
activities related with ethanol: the appetitive behaviours that are those related with the obtaining of the
drug (search behaviour) and the consumption processes that control the quantity of ethanol consumed
once its consumption has begun.

In this model, the animal is conditioned to different concentration of alcohol as a support. Conditioning
boxes are administrated daily in which the animal has to press a lever to obtain 20 minutes to access to
the drug. During the search period, the animal cannot consumption behaviours and once it accesses to the
alcohol, it doesn’t need to press more the lever.

To study the strength of the behaviour of the animal for the search of the alcohol, the answer requited
increases along the days and the cut point is set when the animal cannot to complete the activity. If after
that, animals exceed the effort required to access to the alcohol, it seems that they are highly
overstimulated to obtain a substance even when it requires a big effort.

Forced administration models

There are two procedures which combines the procedures of controlled administration by the investigator
and the learning: conditioning to the place and conditioning to the taste.

Conditioning to the place: Rats are trained in a system that allows the presentation of diverse
visual, tactile, olfactory and auditory stimulus in two different compartments. Animals are exposes to
stimulus related with the administration of alcohol (by intraperitoneal injections) and also to stimulus that
are not related with the drug. In this case, animals are place in the system without alcohol administration
and they are free to move in both compartments. It is measured the preference or aversion to the drug by
the time that the animal stays in presence of the stimulus. It is shown that ethanol does not produce
conditioned preference of the place.

Conditioning to the taste: In these studies, the effect of the drugs (non conditioning stimulus) are
related with the ingestion of a new taste of a liquid or solid food (conditioning stimulus). In the test step,
it is evaluated the ingestion or the preference for a taste in the absence of the drug. If it produces
motivating appetites, the consumption or preference will increase and if it produces aversive effects, the
ingestion or the preference will decrease.

STUDIES WITH RABBITS

In the Institute of Basic Sciences and Preclinical of Girón “Victoria de Girón”, different investigators
studied the variables of the oxidative stress in rabbits treated with ethanol and hypercholesteraemic diet.

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For the study, they used 20 rabbits, race New Zeland, male, 100 days of live. Rabbits were divided into
four groups with the same size: treated with ethanol, treated with hypercholesteraemic diet, treated with
ethanol and diet and a control group.

The assignment of each one to the groups were done by a schedule of random numbers.

It was used ethanol 40% diluted in water (3g/Kg body weigh) in just one daily dose for a period of 16
weeks administrated by oral way. In the end of the treatment, blood was extracted to make the
determinations on it of the parameters that indicate the oxidative stress. The variables superoxide
dismutase (SOD), catalases (CAT) and malonil-dialdehyde (MDA) were analysed.

Rabbits treated with diet and ethanol show values of CAT and MDA higher than rabbits treated with diet.
As a conclusion, the chronic consumption of alcohol, although in moderated doses, alter the antioxidant
system, so it cannot be affirmed that alcohol has a protective effect against the atherosclerosis and that the
consumption of a diet rich in cholesterol increases de lipid peroxidation and the enzymes SOD and CAT.

PHARMACOLOGICAL INTERACTIONS

A pharmacological interaction consists on a modification of the effect of a medicine caused by the

administration of other medicine, medicine plants, food or drinks at the same time what causes a vary in
the therapeutic effect or the appearance of a different effect.

Depending on the mechanism of appearance, the pharmacological interactions can be classified in

pharmacokinetic interactions that produce an alteration in the transport of the medicine across the body
affecting the processes of absorption, distribution, metabolism or excretion and in pharmacodynamics
interactions that affect the mechanism of the medicine because modify the union between the medicine
and the receptor.

Interactions of the medicines with the ethanol

The interactions of the medicines with the ethanol can be pharmacokinetics and pharmacodynamics, but
in most of the cases the effect observed is a combination of both types of interactions.

Anti-inflammatory no steroid

 “Paracetamol”: ethanol increases the metabolism of the “paracetamol” what increase the hepatic
damage.
 “Ácido acetilsalicílico”: this medicine increases the absorption of ethanol in the thin intestine.

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  “Ibuprofeno”: increases the risk of intestinal bleeding.

Antibiotics

 Penicillin G: the ethanol reduces the therapeutic effect of the penicillin

Antihistamine

 Difenhidramina, clorfenamina: the ethanol increases the effect over de central nervous system:
somnolence, sedation and reduces the motor abilities.

Muscular relaxant

 Carisoprodol, ciclobenzaprina: the ethanol increases the sedative effects.

Effect of the ethanol over the medicines

The main pharmacokinetics interactions caused by the ingestion of ethanol with medicines are:

Inhibition of the metabolism of the medicine when the ethanol is occasionally ingested.

Induction of the metabolism of the medicines in chronic consumers. In this case, the interaction of the
ethanol with paracetamol or ácido acetilsalicilico, can cause an increased in the hepatotoxicity because
the metabolites of the first medicine increases and because the irritant effects of the gastric mucus
increases too with the second medicine.

Regarding to the pharmacodynamics interactions are the most frequents. This type of interactions appears
when ethanol is consumed with medicines that affect the central nervous system.

Effect of the medicines over the ethanol

When ethanol is consumed via oral, it is absorbed in the intestine and then goes through the bloodstream
along all the organism. The metabolism of the ethanol is carry out by the enzyme alcohol dehydrogenase,
which oxides the ethanol to acetaldehyde that is a toxic compound that causes the adverse effects of the
ingestion of alcohol.

The disulfiram is a medicine that inhibit the aldehyde dehydrogenase that oxides the acetaldehyde to
acetic acid. This inhibition provokes an increase of the acetaldehyde concentration what causes cephalea,
nauseas, unclear vision, shaking, among other symptoms. These symptoms have as a consequence that the
patient doesn’t want to consume alcohol.

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 DISADVANTAGES & BENEFITS

When ethanol is consumed, it can interfere with other substances or compounds on our body and it can
generate different results:

To treat the ethylic intoxication there are not methods to eliminate the toxin from the body. Gastric
lavage is not useful because the ethanol is quickly absorbs; activated carbon has not any effect with the
ethanol; and other methods (ex.: RO15-4513) just helps to reduce the symptoms that it produces but not
to eliminate it. While the ethanol concentration is higher, the symptoms will get worse and can cause
death in extremes cases. (J. Roldán, 2003)

It interfere in different lab tests such as determinations of cholesterol, corticoids, triglycerides,

glucose, lactate, uric acid…

It intensifies the toxicity of other toxins (ethionamide, guanetidine, nitro-glycerine, sulfonylureas,

biguanides, salicylates…). (Martín, 2014)

Ethanol can also disrupt the personality of the one who is taking it:

Loss of the memory, depression and violent reactions that can affect the daily life. Alcohol is
consistently associated with violent crime, it could be because the effect which has in brain receptors
might reduce fear and anxiety about the social, physical, or legal consequences of one’s actions. It also
affects cognitive functioning, leading to impaired problem solving in conflict situations, and overly
emotional responses or emotional lability. (Robin Room, 2005)

Ethanol intake during long periods of time can cause severe illness:

Excessive alcohol consumption is associated with reproductive disorders. Ethanol involve alterations
in critical hormonal factors controlling reproductive functions but also due to the toxic action of its
metabolites in the organs that constitute the reproductive systems of both sexes. In pregnant women,
ethanol can produce abortion, premature delivery or fetal alcohol syndrome. Fetal Alcohol Syndrome is
diagnosed by three criteria: Deficiency in the growth (height, weight or both), specific facial features
related with skeletal deformities and cardiac problems, and disorders in the Central Nervous System.
(Leandro Néstor Quintans, 2013)

It causes dependence, because of this, alcoholic do not follow a balance diet and this can origin
malnutrition. Primary malnutrition occurs when alcohol replace other nutrients in the diet which are not
consume. Secondary malnutrition happens when ethanol interferes in the absorption of the nutrients
although they are enough. (Cortés, 2008)

Some types of cancer are related to ethanol consumption such as throat and mouth, breast… Other
organs like liver, stomach and pancreas can be also affect.

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 The immune system can be weakest, that would reduce the resistance to infections, increasing the risk
of pneumonia, heart failure… (Monteiro, 2001)

Although all of these, ethanol can be used as an advantage:

It interfere in the methanol and ethylene glycol oxidation, so it is use in severe intoxications of
these compounds. Ethanol do not let formaldehyde and formic acid to be formed, these are the
metabolites which cause the toxicity of methanol. Ethanol has higher affinity to alcohol dehydrogenase
than methanol so it joins to the enzyme and avoid the toxicity of the methanol. It has to be administrated
about 100-150mg/100mL in concentration of 20-30% if it is administrated orally or 5-10% if it is
intravenous. The ethanol has to be given when the methanol concentration is under 20mg/100mL or the
pH of the patient is lower than 7,3. In intoxications with ethylene glycol, ethanol does the same that with
methanol, we can use ethanol treatments when more than 50mL or 2g/L of ethylene glycol are intake. (J.
Roldán, 2003)

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