22 - Tox effect on Skin

Question 1

Two major layers of skin

Answer 1

1. Outer epidermis
2. Underlying dermis
   Seperated by basement membrane

Question 2

Components within skin

Answer 2

Collagen and elastin secrete fibroblasts (strength + elasticity)
* Keratinocytes ➔ corneocytes (fill with lipid, 80% keratin)
* Stratum corneum: outermost, “dead”

Question 3

Percutaneous absorption

Answer 3

Uninjured stratum corneum great barrier
* Uptake of hydrophillic mol. ↑ during water immersion
* Chem uptake can be sig.

Question 4

Skin uptakes can be predicted using many formulas

Answer 4

logP and Mw
* Log Kp = C1 - C2(Mw) + C3(logP)
* C are constants

Question 6

Agents with percutaneous systemic toxicity

Answer 6

1. OP
2. Lead tetraethyl
3. Formamides -reproductive tox
4. Carcinogens

Question 7

What are the two kinds of contact dermatitis?

Answer 7

Both involve inflammation, redness, thickening, scaling, blisters
1. Direct contact irritation
2. Allergic contact dermatitis

Question 8

Direct contact irritation

Answer 8

80%
* not specific to person
* Corrosive, acute and chronic cumulative
* Easy to replicate in animal models

Question 9

Allergic contact dermatitis

Answer 9

Involves immune system
* Complex biomolecular interactions
* Hard to predict

Question 10

Test for irritant dermatitis

Answer 10

Draize test
1. test article applied to intact skin
2. 24h patch removed
3. Rate endpoints (erythema, eschar, edema)

Question 11

Examples of occupational skin toxicity

Answer 11

1. Allergic contact dermatitis to dichromate;
2. Phototoxicity from lime juice

Question 12

Sensitization Phase (Induction Phase)

Answer 12

1. Sensitising agent (or metabolite) interacts w/ skin proteins
2. Form antigenic compelxes (Hapton-protein)
3. Langerhans cell capture complexes ➔ migrate to local lymph nodes
4. Memory T cells

Question 13

Subsequent Exposure (Elicitation Phase)

Answer 13

Upon re-exposure, memory T-cells initiate an immune response ➔
* Inflamm.
* Allergic contact dermatitis

Question 14

Skin Sensitisation Adverse Outcome Pathway

Answer 14

There are 4 key events in development of skin sensitisation

Question 15

KE1

Answer 15

Molecular Initiating Event:
* Formation of stable hapten-protein complexes
* Sensitising agent must be naturally electrophilic or converted

Question 16

KE2

Answer 16

Stim. of local inflamm. response,
* Involve keratinocytes ➔ elicitation of danger signals and cofactors promoting adaptive immunity (e.g. Nrf2-Keap1-ARE pathway)

Question 17

KE3

Answer 17

Activation of dendritic cells (Langerhans cells)

Question 18

KE4

Answer 18

Activation, division and differentiation of T lymphocytes

Question 19

Human Repeat Insult Patch Test (hRIPT)

Answer 19

Assesses irritation potential after repeated application
* Expensive, time consuming
* Risky
* Patch removed after 24h, response recorded 48h
* Repeated 9x

Question 20

hRIPT Challenge Phase

Answer 20

After rest period (10-21d) patch applied to naive site to challenge for sensitisation
* Same duration

Question 21

Three levels of challenge used in hRIPT

Answer 21

1. Open patching (no cover)
2. Semi-occlusive patching (2 sides open)
3. Closed patching (4 sides sealed)

Question 22

Local Lymph Node Assay (LLNA)

Answer 22

Proliferation of draining lymph node cells (LNC) induced by topical exposure to article
* Correlates with extent of skin sensitisation
* Use radiotracer

Question 23

KeratinoSensTM

Answer 23

Non-animal Detection of KE2
* Human keratinocytes stably harbouring luciferase reporter gene under control of ARE pathway
* Activity of Nrf2 transcription factor measured through luminescence detection

Question 24

Keap1-Nrf2-ARE regulatory pathway

Answer 24

Regulator of cyto-protective responses to electrophile and oxidative stress
* Control expression of detox, antioxidant + stress enzymes and proteins

Question 25

HCLAT: Human Cell Line Activation Test

Answer 25

**Non**-animal *investigation* of **KE3** * Test *applied* to human **monocytic leukemia** cell line (THP-1) (*surrogate* for **dendritic**) * **Activation** detected via modulation of **CD membrane** *markers*

Question 26

Direct Peptide Reactivity Assay (DPRA)

Answer 26

**Non**-animal in *chemico* assay whIch *models* **KE1** * Use *synthetic* chem containing **lysine** or **cysteine** * **Incubated** with *test* ➔ measure how much **synth.** **left** ➔ ***correlate*** with **sensitisation** potench.

Question 27

Cross Reactivity

Answer 27

When mol. *share* same **functional** groups, and a particular one has *provoked* **sensitisation** earlier * Several **amines** produce this

Question 28

Drug Induced Skin Sensitisation

Answer 28

**Penicillin** antibiotics * ***Haptenation*** reaction * ***Rashes*** * Drug reaction with **Eosinophilia** and **Systemic** Symptoms (***DRESS***)

Question 29

Azo based dyes and pigments

Answer 29

Azo***reductases*** in **microflora** can *metabolise* and ***split*** azo bond ➔ **2** ***amino*** groups * These *reductases* present in **skin** * ***Sensitisation*** + ***geno***tox

Question 30

Phototoxicity

Answer 30

Chemicals often *abs* **UVA** light ➔ **excited** and *transfer* energy to **oxygen** * **Skin** becomes ***red*** and ***blister***ed

Question 31

Phototoxic Drugs

Answer 31

1. **Oral**: *Tetracyclines*, fluoroquinolones, *NSAIDs* 2. **Topical**: Tetracycline *ointment*, *retinoid* drugs

Question 32

Phototoxicity Tests:

Answer 32

1. **Neutral Red Uptake** Assay: 3T3 mouse **fibroblast** cells *exposed* to **test** article in presence of ***UVA*** 2. **DPRA-UV**: DPRA assay (see earlier) but with ***UVA*** light source 3. In **vivo** (e.g (Nude rb/rnu) rat)