Lezioni, Tossicologia marina

Marine toxicology

Toxicology

Toxicology is the science that studies the harmful interaction between toxins and biological systems. In particular, toxicology studies the natural effects and evaluates the probability of these effects (risk evaluation). It also studies poisons, which are substances that have a toxic and dangerous effect on a living system. These can be natural, like plant alkaloids (e.g., cocaine, morphine) or a nerve gas.

We define a substance as a poison on account of its use. In fact, penicillin is a poison just for bacteria and not for humans, who don't have the cell wall, the penicillin target. So the toxicity depends on the target of the xenobiotic and the amount that you hand out. DDT (dichlorodiphenyl-trichloroethane) remains in the environment because it is very lipophilic; it has been forbidden since the 70s. DDT takes place in adipose tissue, where it is accumulated. It does not represent a problem until lactation when milk passes from the mother to the newborn.

Xenobiotics undergo several transformations in our body. Most of the time, they are easily removed, but it can happen that a non-toxic substance becomes toxic. The main 'transformation center' is the liver. Pirazolidin alkaloids are metabolized in the liver, and they are eliminated easily. If fatty acids envelop these molecules, esterase cannot act, and Cytochrome p450 intervenes, which generates a pyrrole. Alkylating agents interfere with DNA replication and nucleophilic groups (-SH) on proteins, causing several damages; they are, in fact, promutagenic agents. Seventy-five percent of xenobiotics become more hydrophilic after transformation processes and are thus easily removable.

History of toxicology

Toxicology was born with human beings: already ancient people had to take care of women, kill animals to eat, and deal with plant and animal poisons. Toxicology derives from the Greek term toxicon, initially used to refer to curare, used for hunting animals with arrows. It is poisonous only if in contact with blood, not if you ingest it.

In ancient Egyptian papyri, there is a lot of information on rising toxicology: Eber's papyrus, dated 1552 BC, contained a lot of details on poisons, for instance, the alkaloid of belladonna (from Atropa belladonna) and opium. Ancient Greeks had professional knowledge and used it in many ways, including for political purposes. Hippocrates described a lot of poisons, toxicological principles, and overdoses of toxins, indicating that they knew the exact amount of poison to give a person. Poisons were common in ancient Greece and were studied to develop antidotes.

Nicander, the physician of Attalo, was allowed to use poisons on prisoners to study the metabolic effect. The results were collected in a book. He identified 22 specific poisons (mainly from Reptilia, like lidoxyde, aconite, opium). They knew basic antidotes, like making tea with linen seeds to induce vomiting, cleaning the gastric system, and understanding the benefits of removing infected blood and venom from bites.

Mithridates used experiments on prisoners to find antidotes to poisons and toxins. Legend says he drank a mixture of 50 different antidotes to protect himself. When his city was assaulted, he tried to kill himself by drinking a large amount of poison, but he was already immune and couldn’t succeed.

In ancient Rome, politicians frequently faced problems and used toxins to kill people for political and personal reasons. Poisoning was so common in 82 B.C. that Silla promoted a law against poisoning. Diascoride, Nero’s physician, classified every known poison with pictures and descriptions. He divided poisons into three categories: vegetal, animal, and mineral. This classification is still used today.

Modern toxicology

In the Middle Ages, poisoning was an art, especially in Italy, France, the Netherlands, and the UK. During the Renaissance, the art of poisoning reached its peak. In Venice, during the council of 10, documented uses of poisons for political targets were recorded, including victim’s names and the costs of murders. Giulia Tofana created Water of Tofana with arsenic and other powders, commonly used to kill husbands and enemies.

Caterina De' Medici experimented to find the most powerful toxin for specific situations. She pretended to help the poor and sick but poisoned them to study the metabolic effects to achieve her purposes. She stayed with them until the end to observe every change in the body. Although her methods were unethical, she used a perfect scientific approach.

Paracelsus (1493-1541) built the milestone of toxicology. He focused on chemicals and studied the relationship between chemical structure and toxicity. He was the first to distinguish medical/clinical effects from toxic effects. At that time, many poisons were used as drugs for certain pathologies. For example, Mercury was used to treat sexually transmitted diseases because antibiotics were unavailable. Paracelsus understood that the effect also depends on the dose. All drugs are potential poisons; it depends just on the dose! This is one of the most important principles of toxicology.

Forensic toxicology

Orfila, a Spanish man (1787-1853), discovered the relationship between biological effects and chemical structure. Toxins accumulate in the stomach and then spread to other organs. This was a significant discovery as it initiated forensic analysis to prove murders. It wasn't like today, where they want to know who killed whom; back then, they only wanted to know if a death was due to murder. At that time, it was legal for a rich man to kill a poorer person.

Claude Bernard ('800) characterized the toxicological effect and identified the action site of curare. In 1945, Rudolf Peters studied the mechanism of arsenic gas and found an antidote known as British Anti-Lewisite for soldiers exposed to this gas during the Second World War.

Exposure to xenobiotics

We are exposed to many xenobiotics, which can be harmful or very toxic, in different ways in everyday life. All chemicals are toxic; it depends on the dose and the target. Natural toxins produced by microalgae are toxic and can kill people in a few minutes. Thus, from a toxicological point of view, we must know how to use substances.

Humans are exposed to drugs, food additives, industrial chemical agents, environmental contaminants, and products used at home like cosmetics, detergents, and pesticides throughout their lifetime. There are different kinds of exposure: accidental (involuntary) or voluntary, acute or chronic.

Voluntary exposure was common among children until a few years ago; now, detergent boxes and other potentially toxic items are harder to open for children. Work exposure is also a common voluntary exposure: agreeing to work in an industry or factory means deciding to expose or overexpose oneself to toxic agents.

The Seveso accident caused a lot of pollution leading to a toxic cloud that contained a lot of dioxin. This is an example of accidental exposure. People employed at petrol stations are not only exposed to oil but also to other substances contained in oil, like benzene or lead compounds. Benzene is one of the few substances that is surely carcinogenic for humans. Although it is abundant, exposure can be limited by adopting several precautionary measures.

Exposure can be acute or chronic. People who work at petrol stations have chronic exposure because they are exposed to xenobiotics several times a day, for more days (for a month, for a year, lifetime). Chronic exposure is continuous! Acute exposure is an exposure within 24 hours, even at a high dose.

We can classify substances based on exposure. Is exposure to additives voluntary or accidental? Not all people are conscious of what is in food. Mayonnaise, for example, contains a lot of emulsifiers that combine the two phases. Food additives can be considered both an accidental and voluntary exposure.

ADI - admissible daily intake - is the maximum amount of a substance you can take without having toxic effects. There's a similar parameter for ecosystems. At the moment, toxicology cannot avoid the use of animal experiments; evaluation of toxicity in humans must be tested previously in other animal species.

Toxins vs. toxic substances

What's the difference between toxins and toxic substances? Toxins are natural products, for instance, plant, microbial, and algae toxins; toxic substances are directly or indirectly produced from anthropogenic activities. Some toxic substances can be considered toxins. Toxic substances are just a small part compared to the amount of toxins.

Molds and fungi, in general, can produce large amounts of toxins, even in food like cereals (see aflatoxin). This can become a problem in low-developed countries; all of us must pay attention to what we eat. Natural toxins can be very harmful to humans, even if they come from marine origins. Of course, these marine toxins are not so dangerous for seafood and fish but just for humans.

Ostreopsis ovata blooms lead to the production of large amounts of toxins. The main result is higher mortality for shellfish, sea urchins, and other organisms. Palytoxin can accumulate in sea urchins.

LD50 and toxicity comparison

The value LD50 (lethal dose) allows for the comparison of the toxicity of several xenobiotics, both toxins and toxic substances. Lower values correspond to higher toxicity. Botulin toxin LD50 is equal to 0.00001 mg/kg. LD50 is the dose that causes death in 50% of treated animals after acute exposure (single administration or three administrations in a day). LD50 is measured in mg/kg of body weight. Chemicals like dioxins, for instance, TCDD, can be obtained during chemical processes or from the combustion of a fire. The amounts of these substances in the environment should be checked. Chemistry can build up and mimic just a few molecules; nature can construct many substances that chemistry cannot emulate.

In our bodies, we can detoxify some of these xenobiotics, or not: it depends on the exposure, the dose, and other factors. Selective toxicity. We can have different toxicity in different cell types. Epidermal cells in culture can produce several xenobiotics. It is essential to know how to kill cancer cells and not healthy cells! If you have a melanoma, the best approach would be to find a chemical that acts just on tumor cells. The same applies to pesticides: the objective should be to kill just one species, with high selectivity. However, one of the most discussed issues of pesticides is the effect on non-target species.

Toxicokinetics and toxicodynamics

Toxicological selectivity can be due to two reasons: toxicokinetics or toxicodynamics. Toxicokinetics is the study of the absorption, distribution, metabolism, and elimination of a xenobiotic. Toxicodynamics studies the mechanism of action at the cellular and molecular level (where the xenobiotic acts) and tries to identify microscopic and macroscopic toxic effects.

For instance, insects are more susceptible to DDT than humans. They have a more permeable cuticle than human skin. Smaller areas like those of an insect's envelope can absorb higher amounts of DDT; the difference in this case is in the absorption and not in the mechanism.

The rodenticide norbormide is toxic for rats because their smooth muscle cells have a unique receptor that humans do not have. Other products are toxic to rats because they lack a puking reflex. To avoid changes in rats, they are kept in the same conditions until the experiment. To kill rats, warfarin is normally used: this is an anticoagulant agent that avoids obstruction in blood vessels. It depends on the dose you give to the organism. Penicillin is active against some bacteria because it interferes with the cell wall. Humans don't have cell walls, so due to this selective action, penicillin is not effective on humans.

Dose-response concept

Paracelsus is the father of modern toxicology. He said that all substances are potentially poisons and just the dose can differentiate venom from a healthy substance. So, the dose-response concept is essential in toxicology. Usually, the higher the dose, the higher the toxicity.

The nature of collateral effects can be different:

• Allergic effects;
• Idiosyncratic reaction (reaction that takes place depending on individuals of the same species);
• Immediate or retarded toxicity;
• Reversible or irreversible toxic effects;
• Local or systemic toxicity.

The nature of the response can also differ:

• Death (the extreme pathological effects);
• Pathological lesion (injury, damage);
• Biochemical, chemical, or pharmacological variations.

Increasing the dose can lead to histological effects (e.g., alcoholism and cirrhosis). Biochemical variations can be very harsh, potentially leading to damage to the liver or other organs, with visible wounds.

Types of dose-response relationships

We can discern two types of dose-response relationships. The quantal response, also called 'all or nothing' response, can evaluate a toxic point (yes or no; for instance, died or alive, or got cancer after treatment or not). You have a 'complete answer', just two possibilities. We can identify the percentage of the population that undergoes variation after treatment (for instance cancer, or death, or a particular biochemical state, etc.). The higher the dose, the higher the percentage that undergoes variations.

The gradual response can be measured for example with an enzymatic activity assay. For instance, I want to test the weight shift on my animal model by increasing the dose. There is an increasing effect until the model organism can no longer survive. Thus, the higher the dose, the higher the intensity of toxic effects.

In both cases, we have a dose for which there isn't a measurable effect and a dose with a maximum toxic effect (death). The curve shown in the graph is a sigmoid, built up by interpolating values obtained from the analysis. If you want to represent the LD50 curve, it's better to use the logarithmic way.

LD50 (lethal dose 50) gives you an idea of how powerful the xenobiotic is. LD50 can be obtained from animal experiments; to use as few animals as possible, you can start with a little dose, then increase this dose until it causes death in the animal. Reached this point, treat another animal with an intermediate dose between the last used and the dose that doesn't cause death in the model. Dose is measured with mg/kg of body weight.

Response and concentration

The response is proportional to the concentration of the target;

• The concentration of the target site is correlated to the dose;
• The response is correlated in a 'causal manner' to the administered compound.

Not all substances are completely absorbed by the target, as they can spread on tissues around the target and throughout the body. The target can be a receptor with a specific function (e.g., receptor of arilhydroxycarbonhydrolase. The interaction causes an increase of cyt p450) or a protein (Cyt aa3, hemoglobin. For instance, cyanide interferes with the electron transport. The death in presence of cyanide is really fast because of the lack of oxygen). Receptors are not always involved in toxic reactions.

We can have a toxic response even after an administration, but usually, we see it after more doses. The slope of the dose-response curve can vary depending on the kind of toxic effect and its mechanism. From the curve, we can obtain different parameters, like LD50 (useful for comparison of toxicity), the relative power, the administration way, and the intraspecific variability.

Relative power of a substance

What is the relative power of a substance? The lower the LD50, the higher the relative potency. Ethanol has an LD50 of 10000 mg/kg, instead of dioxin, which has an LD50 of 0.001. Tetrodotoxin present in fugu has an LD50 of 0.01, and botulin toxin has an LD50 of 0.00001.

From the table, we can deduce that substances have different LD50 depending on the administration way. Pentobarbital is more toxic if administered intravenously, and it is the same for DFP (di-isopropyl fluoro phosphate) and procaine (anesthetic). For isoniazide, LD50 is almost the same for all the routes of administration.

LD50 can differ among closely related species (see the table with rodent species). Many substances can cause toxicity in different organs depending on the species on which the xenobiotic is tested. Ipomeanol is effective in the lungs of all the species shown in the table; it can also be toxic for a mouse's kidney and a hamster's liver. Thus, the location of tissue damage is relative to the species.

Toxicological experiments

In a toxicological experiment, other parameters must be considered:

• ED50 → efficient dose in 50% of treated animals.
• TD50 → toxic dose in 50% of treated animals.

Logically, we have first the ED50, then TD50, and finally LD50. The proximity of TD50 and ED50 indicates the safety margin of the substances. The therapeutic index TI can be obtained from the ratio LD50/ED50 or TD50/ED50. If the TI value is high, it means that LD50 is very high compared to ED50, so therapeutic effects are not related to toxic effects. For a drug, the safety margin can be defined as the ratio between the LD1 → lethal dose on 1% of the studied biological system and ED99 → efficient dose on 99% of the biological system. TI doesn't provide information about the overlap between toxic effects and therapeutic effects, which can be extrapolated from the dose-response curve. The safety margin in the plot is represented as the gap between ED99 of therapeutic effect and LD1 of toxic effect.

Threshold dose and NOAEL

For some kinds of toxic effects and compounds, there is a threshold dose. Under that dose, there is no effect in any individual of the population treated (the effect is too low and cannot be measured). With NOAEL – No Observed Adverse Effect Level – we indicate the dose at which the effect is not measurable. The evaluation of NOAEL is based on toxicity tests in animals, using the most sensitive species and the most fitting test. For some compounds, like carcinogenic ones, there is no NOAEL. Having the NOAEL value means the possibility to determine the exposure limit.

The ratio between NOAEL and SF (Safety Factor) gives us the ADI, Admissible Daily Intake, also measured in mg/kg of body weight. We have the ADI and TDI (Admissible and Tolerable Daily Intake). TDI indicates the amount of a toxic compound that can be taken over a lifetime without effects. TDI and ADI are conceptually the same, but the first is used in environmental toxicology.