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Toxicology > Dermal toxicology > Flashcards

Dermal toxicology Flashcards

1
Q

What is the function of skin

A
  • Barrier – protective cushion against the environment
  • Controls water loss – 15 ml/h
  • Temperature regulation (Heat loss – vasoconstriction, dilatation, Sweating – up to 5 l/h with exercise)
  • Sensation – touch, pain receptors
  • Controls chemical entry
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2
Q

What is the epidermis and what cells are present

A
  • Basal layer (keratinocytes, metabolism)
  • Cell differentiation – increasing keratinisation
    (Stratum spinosum, Stratum granulosum, Stratum corneum)
  • Langerhans cells – antigen presenting
    “patrol” the spaces between viable keratinocytes
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3
Q

What is the stratum corneum

A

the physical barrier = outermost layer of the epidermis
several layers of highly keratinised, dead cells (enucleated, compressed cytoskeleton)
Endpoint of epidermal differentiation
- Constantly renewed and shed -

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4
Q

explain the bricks and mortar model

A

Comeocyte ‘bricks’ and lipid ‘mortar’ make up the skin ‘wall’. Topically applied chemcials can either penitrated straight through both bricks and motor (transcellular) or move via the lipid ‘mortar’ avoiding the ‘bricks’ (intercellular)

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5
Q

what makes up the lipids in the skin

A

Ceramides

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6
Q

Describe absorption of chemicals through the skin

A
  • Stratum corneum is the major barrier. Absorbed material maybe removed in dermal blood supply, though skin is not highly perfused compared to lung, liver, kidney.
  • Large S.A. 18000 cm2 + Permeability varies with site (Axilla>scalp>forehead>back>abdomen)
  • Damage increases permeability
  • Species differences
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7
Q

what is percutaneous absorption

A

one major factor influencing absorption of a particular chemical: lipophilicity - LogP/LogKow
= [ ]oct/ [ ]water. Anything >3 is lipophilic (testosterine = 3.32)

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8
Q

what happens when highly lipophilic chemicals enters stratum corneum

A

hard time leaving it
- viable epidermis is aqueous

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9
Q

what are the other influencing factors of skin absorption

A

Form of deposition (Liquid»solid>gas), Concentration, Temperature/ humidity/ occlusion. Anatomical site, Damage, Metabolism/binding

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10
Q

what has in vivo studies using human volunteers shown about percutaneous absorption

A
  • Very expensive, Difficult to obtain ethical approval,
    Difficult to conduct studies
  • Biological monitoring required – results require careful
    interpretation
  • Still regarded as the “Gold Standard” by many
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11
Q

what has in vivo studies using experimental animals shown about percutaneous absorption

A
  • Also expensive
  • Ethical approval easier but still contentious
  • Difficult to conduct studies
  • Biological monitoring required – results require careful interpretation
  • Traditional animal models have more permeable skin – domestic pig best
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12
Q

List the In vitro models systems -

A

diffusion cells, perfusion skin flap
- Animal skin (same issues as in vivo)
- Human skin (ethics and expense)
- Human skin equivalents
- Full thickness, dermatomed, epidermal membranes
- Easier to conduct and interpret
- Standardisation, guidelines (OECD, EPA)

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13
Q

what is a finite dose

A

“real life” exposure such as splash or brief immersion, cosmetics, topical medicines etc = Absorption is limited:
by dose (which can become exhausted as the compound is absorbed) or By vehicle (which may evaporate – no vehicle means no concentration gradient)

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14
Q

How is absorption effected during finite dosing

A

Absorption rate changes constantly as a result of the processes
- Exposure time may vary
-Working day (6 to 10 h depending on the regulatory
regime)
- “Leave on” products – up to 18 h
- “wash off” products – 2 minutes or up to 30 for hair
dyes

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15
Q

what is an infinite dose

A

Not representative of “real life” (except maybe a swimming pool), more like a “maximum speed”
Absorption is not limited by dose or vehicle
Absorption rate should reach a steady state after a lag time
Exposure time usually 24 h (sometimes longer)

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16
Q

The terms flux, lag time and permeability coefficient should only be applied to _ doses, when the concentration of the applied dose remains ___

A

infinite
relatively constant with time

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17
Q

How does a finite dose graph on absorption v time

A

dramatic increase then the curve levels off
slightly sigmodial

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18
Q

how can percutanous absorption be predicted

A

In silico = QSAR (knowledge based systems)
[- Log kp = -2.72 + 0.72 Log P – 0.006 MW (r2 = 0.69)
(kp in cm/h)]
Maximum flux Jmax-Kp mutipled by solubility in vehicle
Kp is vehicle specific, Jmax is theorectically independent of vehicle

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19
Q

what is the problems with current in silco approaches

A
  • Predict kp or maximum flux (“infinite dose” parameter, further algorithms needed to predict absorption from a realistic dose)
  • So far, only aqueous or a handful of other, neat, vehicles modelled
  • Do not take into account poor absorption of highly lipophilic chemicals
20
Q

How does the skin metabose xenobiotics

A
  • inactive chemical undergoes functionalisation by (CYP450, Esterases, ADH/ALDH)
  • activated chemicals bind to macromolecules -> toxicity
  • Activated chemicals can then undergo conjugation via transferases to be eliminated
21
Q

what is the CYP450 activity in the skin

A
  • Located in basal keratinocytes, hair follicles, (vasc. Endothelium). Variety of endogenous and xenobiotic substrates
  • Present at low activities when expressed per mg tissue (1-5% of liver, except 2S1)
  • Little effect on absorption but can cause toxification (e.g. diol epoxide formation)
22
Q

Given examples of CYP450s in the skin

A
  • CYP 1A1 – aryl hydrocarbon hydroxylase, 7-ethoxyresorufin de-ethylase, all species
    (Basal levels in humans very low but induced by tobacco smoking)
  • CYP 1A2 [mouse CYP 1B1] mRNA in humans
  • 2A, 2B, 2E1, 3A4, 3A5 activities, proteins or mRNA found (not in all species)
  • 2S1 more highly expressed in skin than liver (An endogenous role in retinoic acid metabolism)
22
Q

describe esterases activity in the skin

A
  • Cytosolic and microsomal
  • present throughout epidermis and dermis
  • can influence absorption by reducing lipophilicity – - -exploited for transdermal delivery of ester pro-drugs
    [fluazifop butyl, umbelliferyl esters, parabens]
23
Q

Describe Alcohol and aldehyde dehydrogenases activity in the skin

A
  • ethanol, ethoxylates, cinnamic alcohol and cinnamaldehyde
  • may influence toxicity = role in skin sensitisation
24
Q

Describe transferases activity in the skin. Describe each enzyme and which drug they work on

A
  • detoxifys with a higher activity than P450
    affect availability but not percutaneous absorption
  • Sulphotransferases (triclosan, minoxidil)
  • Glucuronyl transferases (microsomal) (triclosan)
  • N-acetyl transferases (cytosolic) – aromatic amines
  • Glutathione transferases – Free radicals, electrophiles e.g. Dinitro chlorobenzene
25
Q

describe NAD(P)H quinone reductase activity in the skin

A

activity is similar to liver
-detoxify quinones (convert to hydroquinones), prevent oxidative stress

26
Q

what is the symptomology of local topical toxicity

A

Corrosion, ulceration, contact urticarial, physical dermatitis, irritation, immune reactions, Cancer (UV/chemical exposure)

27
Q

dermal absorption can lead to _
Give an example

A

Systemic toxicology
chloracne

28
Q

describe Dermatitis

A

Irritant contact dermatitic = 70-80% of human contact dermatitis
- erythema, scaling, thickening
- many possible causative routes by many agents
(doesnt require previous exposure, direct damage to cells = release of cytokines, local inflammation
Can be delayed or immediate, acute or cumulative/chronic)

29
Q

we still have little knowledge of mechanism of irritation - surfactants are best researched, describe there mechanism -

A
  • Stimulate IL-1a release – this stimulates release of IL6 and IL8, Phospholipase-a2, and TNFa.
  • These lead to symptoms via cellular changes.
30
Q

non-membrane damage irritants dont appear to work soley via release of _ what might by why ?

A

IL-1a
Maybe ROS mechanisms

31
Q

describe the mechanism for irritant contact dermatisis when JP-8 is absorbed

A
  • activates both IL-1a and causes oxidative/osmotic/membrane/mitochondria stress
  • this causes IL-6 signaling, P38 MAPK signalling and ERK/MAPK signalling
  • these 3 pathways cause vasodilation & Extravasation, apoptosis, cell growth & proliferation
32
Q

what is allergic contact dermatitis

A

10% of human contact dermatitis
- IgE mediated, 2 phase response
Caused by Latex, DNCB and amine hair dyes

33
Q

Describe the induction phase of allergic contact dermatitis

A
  • Chemical is absorbed, binds to proteins on Langerhans cells (complete allergen)
  • Damage to keratinocytes – release of IL-6, IL-1B
  • Direct allergen to lymph node, interacts with t-lymphocytes
  • Replication of sensitised to T-lymphocytes in circulation
34
Q

describe the elicitation phase

A

allergen reacts with circulating sensitised T-lymphocytes when allergen enter viable epidermis

35
Q

how does Haptens (DNCB) and prohaptens

A

prehaptens are activated before entering the stratum corneum, prohaptens are activated after.
-haptens then interact with protein and produces immunogens

36
Q

What are the test for skin sensitisers

A
  • Murine local lymph node Assay = in vivo refinement method, measures the sensitisation phase (DNA synthesis in draining lymph nodes), no longer permissible for new cosmetics
  • More recently developed approaches = peptide reactivity, activation of keratinocytes and dendritic cells
37
Q

Explain the direct peptide reactivity assay

A

“In Tubo” test
- Measures depletion of model peptides containing nucleophilic residues
- Test chemical incubated with synthetic peptides containing cysteine (10:1 ratio) or lysine (50:1) ratio (one concentration, 24 h period)
- Analysis by HPLC UV and/or LCMSMS
- % depletion relative to vehicle control - classification

38
Q

Why is it difficult to test prohaptens using direct peptide reactivity assay

A

Prohaptens are difficult to detect as there are no cells, therefore no enzymes.
Pretreatment with peroxidases has been proposed as an extension, but some say this may overestimate detection of prohaptens

39
Q

Describe the keratinosens assay

A
  • Kelch-like ECH-associated protein 1 (Keap 1) is an intracellular sensor of reactivity.
  • Covalent cross linking of Keap 1 results in dissociation of Nrf2
  • Results in expression of various genes involved in response to sensitisers.
40
Q

what is the Nrf2Keap protein

A
  • The Nrf2keap protein is an intracellular “sensor” of electrophilic chemicals and reactive oxygen species.
  • In the absence of such chemicals/species, the SH groups on Nrf2keap remain as SH groups.
  • If Nrf2 becomes detached from keap, it is ubiquitinated and “tagged for destruction” by the proteasome.
41
Q

If there are electrophiles or ROS present during the keratinosens assay

A
  • Then the SH groups either react with chemicals or become cross linked.
  • This results in the release of Nrf2 without ubiquitination.
  • Nrf2 translocates to the nucleus and binds (with Maf) to the antioxidant response element (ARE), leading to transcription of important genes in the response to chemical/ROS effects
42
Q

What is the mechansim of KeratinoSens assay

A

Reporter cell line for Nrf2-pathway - A HaCat (immortalised keratinocyte) cell line in transfected with a construct containing the ARE, a SV40 promoter and luciferase gene
- If there is no electrophilic chemical or other reactive agent, Keap1 and Nrf2 remain associated, and Nrf2 does not enter the nucleus, so there is no transcription of luciferase.
- If an electrophile is present in the cell, the SH groups are no longer present; this triggers dissociation of Nrf2 from Keap1; Nrf2 can then bind to the ARE and the luciferase gene is transcribed. When ATP and luciferin are present, luciferase will convert these to photons of visible light, which are measured in a luminometer.
- A range of concentrations of test chemical are measured; the concentration which results in a three fold increase in the background luminescence is reported.

43
Q

the effect of the test compound on cell viability is also measured during the KeratinoSens Assay

A

If the cell is dead, there will be no luminescence, so it is essential that the test compound does not result in a significant decrease in cell viability.

44
Q

T/F KeratinoSens assay can detect ‘prohaptens’

A

True
Though the metabolic competence of HaCats is nowhere near as good as skin S9 fraction.

45
Q

h-CLAT assay - explain how they work

A
  • Monitors expression of CD86 and CD54 on the surface of THP-1 cells (monocytic leukaemia cell line)
    = Expression of these marker proteins is “precursor for migration to lymph nodes”
    = Obtaining langerhans cells is more difficult
    = CD86 expression is a good marker of allergens (as opposed to irritants)
    = Cells are exposed to the test chemical for 24 h, then treated with fluorescent labelled antibodies to CD86/CD54 (flow cytometry).

Toxicology (11 decks)

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