Posts Tagged ‘ toxicity ’

Comparative Nitrogen Excretion

Introduction

In animals all waste products must be excreted from the body in some manner. The urinary system is involved in the excretion of nitrogenous waste products such as urea, uric acid or ammonia. The excretion of nitrogenous waste from animals is important because if it is allowed to build up, it can prove toxic. We obtain nitrogen from protein in the diet, when protein is metabolised it produces amino groups which are able to form the highly toxic ammonia. Different classes and species of animals deal with toxic ammonia in different ways. A summary:

Compound Chemical Formula Toxicity Water Solubility Animals
Ammonia NH3 High High Aquatic Animals
Urea (NH2)2CO Moderate Moderate Terrestrial Animals
Uric Acid C5H4N4O3 Low Low Reptiles and Birds

Method of Excretion by Species:

Fish

Fish excrete nitrogenous waste as ammonia; this is unusual because ammonia is highly toxic therefore storage in the body can pose a risk. However, fish are able to cope due to their environment – the large volumes of water they reside in allow them to continuously excrete ammonia (without the need for storage) directly into the water, diluting the ammonia to non-toxic levels.

Fish deaminate (remove the amine group) amino acids (obtained in the diet by protein consumption) in the gills. The pure ammonia diffuses into the respiratory water which is leaving via the gills; it is therefore excreted at the site of production with minimal time in the body to reduce toxicity.

There are two extremes of environment which fish have to adapt to however – marine and freshwater. The difference in concentration of salt between these two environments has led to slightly different methods of dealing with nitrogenous waste. Osmoregulation of fish living in fresh water requires them to excrete large volumes of dilute urine. This is due to osmosis, which allows large volume of water to enter body fluid from the surrounding hypotonic freshwater.

The converse is true for marine fish, the hypertonic water means water leaves bodily fluid by passive osmosis, in an attempt to conserve water small amounts of concentrated urine are released.

Elasmobranches, such as sharks, primarily live in seawater and are able to produce urea as well as ammonia. This is important for osmoregulation, as said earlier the hypertonic seawater means water is able to leave bodily fluids by osmosis. By producing urea (which is less toxic than ammonia and therefore storable), elasmobranches are able to increase their osmolarity higher than the seawater and therefore take up water from the sea like freshwater species. This is due to the urea being retained in the blood and therefore increasing the osmolarity of the blood.

Fish still have kidneys however, despite nitrogen excretion being handled by the gills. Instead the kidneys are involved in the excretion of excess water and divalent ions, e.g. Mg2+ and SO42-.

Lungfish, which are able to live out of water, have another adaption. They excrete ammonia in the usual way when in the water, but during times of drought when the lungfish buries itself in the mud the production of toxic ammonia could prove fatal. So instead, when buried in the mud, the lungfish produces urea which can be accumulated and excreted into the environment at less toxic levels.

Amphibians

As with fish the type of nitrogenous excretion depends entirely on environment. Typically tadpoles use the same method as fish – ammonia excretion via the gills. However adult amphibians produce urea as they do not remain constantly in the water. The urea is stored, diluted in the bladder, but as amphibians may be prone to desiccation they have adapted the ability to reabsorb water from the urine when needed. This requires them to have relatively large bladders for storage.

Amphibians do not drink to obtain water; instead they are able to absorb water through a region of the skin called the ventral pelvic area.

Birds

All birds excrete a paste like substance called uric acid (which contains the waste nitrogen) independent of habitat. It initially begins as watery urine which travels down the ureters, from the kidney to the cloaca. The cloaca is a unified exit for the gastrointestinal, urinary and reproductive systems.

In the cloaca, the urine mixes with faecal matter from the digestive tract and as much water is removed as possible to produce a dry, crystalline paste containing uric acid. A small amount of watery urine may be excreted as well.

Reptiles

The method of nitrogen excretion depends on the habitat; aquatic reptiles (e.g. turtles) will mainly excrete ammonia (possibly urea) as with fish. Whilst reptiles living in drier conditions (e.g. lizards, snakes & tortoises) excrete uric acid – the low toxicity, crystalline paste. This helps to conserve water and during development in the egg, a build-up is not fatal. Uric acid, as with birds, is excreted from the cloaca.

The kidneys of reptiles do not have loops of Henle and so they are unable to produce concentrated urine also, whilst a large bladder is present in chelonians (turtles) snakes and some lizards do not have a bladder at all.

Mammals

All mammals produce primarily urea (sometime ammonia) which is excreted in urine. Mammals are able to osmoregulate and maximise water conservation by varying the concentration of urine depending on the hydration of the body. Mammals living in environments with plentiful access to fresh water will excrete large amounts of dilute urine. Mammals living in dry or marine environments will excrete small amounts of concentrated urine.

Osmoregulation in mammals is mainly controlled by the hormone ADH, its release (when a mammal is dehydrated) will result in the reduction of water loss and increased reabsorption of water in the kidneys by the loop of Henle.

Osmoregulation

Osmoregulation has been mentioned above in some cases, it can be associated with the excretion of nitrogenous waste because without correct osmoregulation, levels of ammonia can build within the body. A well osmoregulated animal will be taking on water and balancing ions well enough to ensure that nitrogen can be excreted and not build up.

The primary problem facing osmoregulation is the build-up of salt ions, this can occur with animals living in marine habitats.

  • Fish can actively excrete NaCl via gills
  • Some cartilaginous fish such as sharks have specialised salt-excreting glands in their rectum
  • Birds and reptiles have specialised salt-excreting glands on their heads
  • Marine mammals are able to excrete salt in urine efficiently
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Non-Steroidal Anti-inflammatory Drugs (NSAIDs)

Introduction

NSAIDs are non-narcotic analgesics (An analgesic reduces or removes the sensation of pain), they are also anti-pyretic (fever) and anti-inflammatory. These effects are produced by the inhibition of the fatty acid cyclooxygenase (COX) which inhibits prostaglandin synthesis.

Because NSAIDs are non-narcotic they do not cause any largely noticeable effects on the CNS (central nervous system) function. This makes them ineffective against normal nociceptive tests, these are test designed to test pain responses in living organisms and they are specifically used in the testing of new analgesic drugs. Methods usually involve the applying of pressure to a specific point of the organism. NSAIDs only raise the pain threshold when pressure is applied to a swollen and inflamed joint (this is known as analgesia via peripheral mechanisms). Therefore NSAIDs are considered anti-inflammatory agents with a mild central analgesic effect (associated with anti-pyretic action). NSAIDs are therefore primarily used in the treatment of acute or chronic conditions producing mild-moderate pain, especially involving the musculo-skeletal system. Principal utilisation occurs in the horse and dog.

Another benefit of NSAIDs non-narcotic function is that, by having an unnoticeable effect on the CNS they can therefore be used as a pre-med drug, before general anaesthetic, without fear of overloading the CNS. The use of an analgesic before the introduction of pain means that a lower dose will be required when pain is inflicted – thus reducing the chances of side effects associated with high doses. However, the type of NSAID must be selected for carefully as there may be a possibility of renal damage/toxicity. There are only a couple of NSAIDs which are believed to be renal safe.

Prostaglandins and Inflammation

NSAIDs work by inhibiting prostaglandin synthesis by targeting the COX enzyme. Prostaglandins activate the inflammatory response giving the production of pain and fever, they are produced when leukocytes reach a site of damaged tissue in an attempt to minimise tissue destruction.

Prostaglandins are involved in several other organs such as the gastrointestinal tract (inhibit acid synthesis and increase secretion of protective mucus), increase blood flow in kidneys, and leukotrienes which promote constriction of bronchi associated with asthma.

Question:

What is/are the precursor(s) of prostaglandins?

The answer may be found in the ‘Mechanisms of NSAID Action’ section.

Cyclooxygenase Enzymes (COX)

COX forms two isoforms, COX-1 and COX-2:

  • COX-1 is often thought of as being the ‘good’ COX; this is due to its involvement in tissue homeostasis. It is required to keep the body ‘normal’ – primarily the synthesis of prostaglandins responsible for protection of the stomach lining. (Constitutive physiological).
  • COX-2 therefore is thought of as the ‘bad’ COX; this is because it is produced during inflammation, by the inflammatory cells which have been activated by cytokines. (Inducible physiological).
  • There is some evidence for a COX-3, a possible variation of COX-1 which is also associated with the inflammatory response. It has been found in the CNS and is affected by paracetamol.

Mechanism of NSAID Action

The primary action of NSAIDs is the inhibition of the COX enzyme, by inhibiting this enzyme the production of prostaglandins are also inhibited. The COX enzyme synthesised prostaglandins from fatty acids such as arachidonic acid.

Most NSAIDs inhibit both major forms of the COX enzyme, however all are still considered toxic. Newer drugs which are believed to be COX-2 specific (thereby not affecting the COX-1 enzyme and allowing prostaglandins associated with normal function to continue normal operation) are relatively safer in chronic use. There are fewer side effects which is what makes them be suited to prolonged periods of use. Examples: Merck’s rofecoxib and etoricoxib, Pfizer’s celecoxib and valdecoxib.

The NSAIDs selective for COX-2 are now however under scrutiny, due to reports of cardiovascular toxicity. These include strokes and myocardial infarctions. This has resulted in the withdrawal of certain COX-2 selective drugs such as rofecoxib. Further studies are suggesting that the cardiovascular toxicity of these COX-2 selective NSAIDs may actually not be much greater than ‘trusted’ NSAIDs such as ibuprofen. However, their toxicity still remains a point of research and discussion.

Other Actions of NSAIDs

Besides from the inhibition of the COX enzyme, other actions include:

  • Inhibit superoxides (toxic) and free radicals
  • Inhibit Bradykinin production (A Peptide which dilates blood vessels, lowering blood pressure)
  • Stabilises lysosomes
  • Inhibits metalloproteinases (Proteolytic enzymes whose catalytic mechanism involves a metal)
  • Antagonises interleukin-1 (fever inducer and controlling factor of lymphocytes) and tumour necrosis factor (TNF – cytokine involved in the induction of inflammation and apoptosis, dysfunction of this factor is believed to be involved with the production of cancers.)

IC50

This is the half maximal inhibitory concentration (IC50). It is a measure of the effectiveness of a compound (typically a drug candidate) in inhibiting biological or biochemical function. This quantitative measure indicates how much of a particular drug is needed to inhibit a given biological process (or component of a process, i.e. an enzyme, cell, cell receptor or microorganism) by half. It is commonly used as a measure of antagonist drug potency in pharmacological research.

This can be related to inhibition of COX enzymes, we can use it to find out how many times a dose required to inhibit COX-1 we need to administer in order to inhibit COX-2, i.e. COX-2/COX-1,  a lower ratio is better as it shows that the drug has a higher selectivity for COX-2 the ‘bad’ COX associated with inflammatory responses.

COX-1 Selective               Drugs                       Amount times dose required to inhibit COX-2

  • Aspirin                                                                  170
  • Piroxicam                                                             250

Less Selective COX-1 Drugs

  • Paracetamol                                                            7
  • Ibuprofen                                                                15

Equipotent

  • Naproxen                                                                  1

COX-2 Selective Drugs

  • Meloxicam                                                               <1

The Anti-Pyretic Effect of NSAIDs

NSAIDs do not affect normal body temperature only when pyrexia (fever) is present, do they alter temperature. Bacterial or endogenous (substances from within the body) pyrogens can act directly on the hypothalamus. Heat temperature is regulated in the hypothalamus by controlling temperature regulating peripheral mechanisms such as vasoconstriction/dilation, sweating, shivering and metabolic activity.

Pyrogens activate hypothalamic COX, increasing prostaglandin concentration. The effect of this is that the set body temperature (Average 37oC) is increased (Pyrexia is considered >38oC). NSAIDs counter this by inhibiting prostaglandin synthesis by the inhibition of COX enzymes. By doing this they have effectively blocked the action of the pyrogens on the CNS and return the raised set body temperature back down to normal levels (37oC).

Commonly Used NSAIDs

Some of the most commonly found NSAIDs and their properties:

Aspirin (Acetylsalicylic acid)

Aspirin is a potent anti-inflammatory drug with mild central analgesic and antipyretic actions. It is administered orally and readily absorbed from the stomach and small intestine, an acid drug is well absorbed in an acidic environment. It is metabolised by tissue /plasma esterases. Aspirin may also be used in low doses, daily to prevent platelet aggregation.

In a healthy body, thromboxane and prostacyclin (eicosanoids – fatty acid signalling molecules) are balanced. Aspirin however disrupts this balance in the favour of prostacyclin, inhibiting aggregation.

Aspirin irreversibly binds to platelet COX, however as platelets are produced every few days, the condition is not permanent, and this is why chronic dosing may be necessary.

Aspirin is toxic in cats due to their lack of the enzyme UDP-glucuronyl transferase, therefore when giving aspirin to cats the maximum stated dose is 25mg/kg daily (Compared to 25mg/kg 3-4 times a day in dogs) Toxicity effects may still appear even at lower doses e.g. vomiting, abdominal pain, anorexia and gastric ulceration.

Paracetamol

Paracetamol is a weak anti-inflammatory drug; however it does have a more potent central analgesia and anti-pyretic effects than aspirin (See IC50 table to see a smaller dose is required of paracetamol to inhibit COX-2, the COX enzyme responsible for inflammatory responses).

Due to paracetamol being well tolerated, producing less gastric irritation than aspirin and having much fewer side effects than aspirin, it has become a predominant household analgesic. Acute overdoses can cause fatal hepatic damage; early symptoms include anorexia, vomiting, diarrhoea and abdominal pain. It is the reactive metabolites of paracetamol latching onto -SH groups that cause hepatic toxicity.

The dog is more resistant to paracetamol toxicity than cats, an oral dose is recommended every 6 hours of 25-30mg/kg.

Phenylbutazone

Phenylbutazone is the most widely used NSAID in equine medicine; however it is extremely toxic in humans. It has a long  t1/2 (half-life) of 70 hours and produces severe gastric ulceration and agranulocytosis. It can be administered orally and by intravenous injection. Due to the acidity of the drug, it is readily absorbed from the stomach/duodenum. Phenylbutazone metabolites are weak acids and therefore preferably excreted in alkaline urine. Training horses may have acidic urine, and so it is recommended not to take Phenylbutazone within 8 days of competition.

Half-life in dogs of Phenylbutazone has been recorded at 3-8 hours (however this may vary dependent on the dose). Phenylbutazone can inhibit the synthesis of prostaglandin in inflammatory exudates for 12-24 hours, with the response lasting for up to three days after the final dose in the course.

Signs of Phenylbutazone toxicity include inappetance and depression with weight loss and oedema. The oedema (fluid retention) is due to the decreased NaCl excretion.

Dosing of Phenylbutazone should not exceed:

  • Day 1 – 4.4 mg/kg twice a day
  • Day 2-4 – 2.2 mg/kg twice a day
  • Day >5 – 2.2 mg/kg daily

Phenylbutazone is also administered in combination with other drugs for the treatment of musculoskeletal disorders e.g. Tomanol – Phenylbutazone and Isopyrin

Meclofenamic Acid (Arquel)

Meclofenamic acid is a potent anti-inflammatory, anti-pyretic analgesic. It is more potent than aspirin but similar in effect. As well as inhibiting COX enzymes it has found to be a prostaglandin antagonist, interacting with prostaglandin receptors. It therefore prevents the action of prostaglandin already present possibly exerting a more rapid reduction of inflammation.

Half-life is 6-8 hours; therapeutic levels are maintained with daily doses.

Unlike other NSAIDs onset of Meclofenamic acid is relatively slow, taking 36-96 hours for effects to begin

Prolonged administration in the horse may lead to anorexia, depression, ulceration of buccal mucosa, gastric haemorrhage or diarrhoea

Naproxen

Naproxen is a propionic acid derivative (like ibuprofen or Ketoprofen) with a tendency for reduced frequency of serious side effects at therapeutic doses. In the horse, half-life of the drug is 5 hours; double daily doses have been proven effective in soft-tissue inflammatory conditions such as myositis. In the dog however, half-life is much longer at around 70 hours, it is therefore recommended to avoid this NSAIDs due to excessive toxicity

Ketoprofen

Ketoprofen is a potent COX inhibitor which is also able to stabilise lysosomal membranes and is a Bradykinin antagonist. It is also reported to be an inhibitor of the lipoxygenase enzyme system (iron-containing enzymes which catalyse the dioxygenation [incorporation of two oxygen atoms] of some polyunsaturated fatty acids). It is around 50x more potent than Phenylbutazone, however this is not accompanied by an equivalent increase in toxicity. It is also suggested that it may be cartilage sparing, neither accelerating chondrocyte damage nor reducing proteoglycan production.

Carprofen

Carprofen is a potent anti-inflammatory drug, but is a weak inhibitor of COX. Its mode of action is not yet known but it significantly inhibits neutrophil migration. Due to weak inhibition of COX, toxicity of Carprofen its toxicity tends to be low.

Piroxicam

Piroxicam is a potent and long lasting anti-inflammatory drug. Its half-life of around 60 hours enables lower dosing (alternate days). A higher frequency of dosing will produce standard NSAID toxic effects (see below, section ‘Side Effects of NSAIDs’)

Meloxicam

Meloxicam is a similar drug, but with a shorter half-life (30-40 hours). It is thought to have greater potency for COX-2 than COX-1 therefore side-effects may be less. It is also thought to be chondroprotective (The slowing of degradation of articular cartilage)

Flunixin

Flunixin is a potent versatile anti-inflammatory drug with a short half-life in all species (2-8 hours). However its duration of action is relatively long (24-36 hours)

When to Use NSAIDs

  • Mild to moderate inflammatory lesions and associated pain
  • Acute inflammation and pain
  • Joint inflammation and pain
  • Suppression of pulmonary oedema
  • Endotoxaemia
  • Anti-thrombic

Side Effects of NSAIDs

  • Gastric irritation and ulceration – This is a main side effect of chronic NSAID use, it occurs because when NSAIDs inhibit the COX-1 enzyme, COX-1 synthesises prostaglandins associated with inhibiting acid synthesis and increasing secretion of protective mucus. SO by inhibiting COX-1, the stomach becomes unprotected from the gastric acid causing irritation and possibly ulceration in chronic use.
  • You can protect the gut by administering proton pump inhibitors (Losec), prostaglandin analogues (Misoprostol) or H2 receptor antagonists (Zantac)
  • Vomiting and diarrhoea
  • Hepatotoxicity
  • Renal papillary necrosis, chronic nephritis
  • Bone marrow disturbance
  • Skin rashes
  • Respiratory distress