Development of chronic and passive dosing systems in vitro for genotoxicity

assessment.

Professor Gareth Jenkins, Swansea University, UK

In vitro Toxicology Group, Swansea.

• Longstanding interest in DNA damage and mutation testing.

• Studying the link between mutation and cancer in patients.

• Designing new, more sophisticated in vitro testing strategies for genotoxicity and carcinogenicity.

• Reducing reliance on animals in toxicology.

Genotoxicity • Used as a surrogate for carcinogenicity in

vitro and in short term in vivo tests.

• A 2-year rodent bioassay still also used for carcinogenicity as an apical endpoint.

• Obviously some sectors (e.g. Cosmetics) cannot perform animal tests any longer.

• Tiered approach for genotoxicity employing in vitro and in vivo stages.

Regulatory genotoxicity testing Stage 0: Structure Activity Relationships (SAR), screening tests and physio-chemical properties (of substance and impurities)

Stage 1: Bacterial gene mutation tests (Ames test) Clastogenicity and aneugenicity (in vitro micronucleus assay)

Negative results in all tests

Equivocal result in any test Positive results in any test

Stage 2 (in vivo): Under take one or more of the following recommended assays: 1. Micronucleus assay or chromosome aberration test 2. Transgenic mutation test 3. Comet assay

Substance is not mutagenic

Insufficient evidence to assess the mutagenicity of the substance Review available data and make pragmatic conclusions based on case –by-case study

Positive: if data is robust consider substance to be in vivo somatic cell mutagen and possible germ cell mutagen

Negative after full assessment

Issues with current testing paradigm. • In vitro tests qualitative not quantitative.

• Binary decisions on genotoxicity. Dose response relationships not fully considered.

• Sensitivity not specificity.

• Heavy reliance on animals, used to de-risk misleading positive results in vitro.

• Simple Acute dosing scenarios do not reflect human exposure – lead to misleading positives? Overwhelms cellular defence mechanisms -non-physiological.

The Micronucleus (Mn) assay.

• Standard test for genotoxicity, detects most classes of genotoxins.

• Strict adherence to >50% cell viability, as cytotoxicity is a confounder.

• Usually block cytokinesis and look for Mn in binucleated cells.

• OECD guide available for consistency.

• Automation can increase statistical power enormously.

Thresholds for alkylating agents: A key paper in 2007.

Doak et al., 2007. Cancer Res. 67, 3904-3911.

Cancer Res 2007;67: (8). April 15, 2007

Acute v Chronic (subchronic) in vitro exposures

• Current OECD guidelines recommend acute exposure scenarios for in vitro tests (e.g. 4-6 hours or 24 hours for the Mn test).

• Dose fractionation studies 1970’s and 1980’s done at high dose (100x above LOEL). Mainly focussed on radiation in animal models. Relevance not clear to low dose in vitro situation.

• Human exposure is chronic low dose in many cases.

• Need to better model human exposure scenarios in vitro to assess hazard (and risk).

Subchronic in vitro dosing (TK6 cells)? • We have been exploring the influence of

exposure scenario on genotoxicity. Using simple alkylating agents (MMS, MNU).

• What effect does chronic low dose exposure have on genotoxicity for potent genotoxins.

• Comparison to low dose acute exposure?

• What does this tell us about human exposure situations.

• MMS and MNU delivered at 1/5th the concentration for 5 days or 1/10th the concentration for 10 days.

• Compared to an acute, one-off exposure.

• Each treatment (acute/chronic) 24 hrs.

• High powered studies for Mn induction (large numbers of cells).

• Mononucleate MN assay performed (12,000 cells per dose).

Genotoxicity of chronic MMS and MNU in vitro

Chronic in vitro dosing at low dose level reduces genotoxic hazard.

Mechanisms of action behind the effects.

• DNA repair? Induction of repair enzymes.

• Some evidence for hormetic effect with MNU – induced repair?

• P53 status. Isogenic cells (NH32) deficient in p53 show altered chronic response. Chronic exposure shows same results as acute TK6 system

• Losses of chemical in system, allowing cells to tolerate higher exposures in total.

Chronic dosing effects for Non-genotoxic carcinogens

• Non-genotoxic carcinogens, negative in standard in vitro genotoxicity tests.

• Nickel Chloride shown here.

• But, can show some +ve results under chronic exposure conditions (5 day exposure to 1/5th acute level)

• Linked to ROS induction, which rises daily.

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Passive dosing • An interesting extension to this line of investigation

is the use of passive dosing (PD).

• Here a hydrophobic genotoxin (from an inert polymer source) is passively contributing a low but constant exposure to a cellular system and the mutational endpoints assessed.

• Exposure level can be varied by differential saturation of the loading solution.

• Sampling allows monitoring toxin concentration (or can be estimated from partitioning coefficient).

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Advantages of PD • Constant (low) level exposure to toxin.

• Partitioning coefficient from polymer allows prediction of behaviour.

• More reproducible data as exposure more reliable.

• No toxic co-solvents.

• More realistic exposure scenarios to some toxins.

Smith et al., (2013) Mut. Res. 750: 12-18 Booij et al., (2010) Env. Tox. Chem. Smith et al (2010) Chem. Res. Tox 23: 55-65

Disc delivery system.

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-B[a]P a model hydrophobic genotoxin -MCL5 human lymphoblastoid cells (P450 activity). -Mn (mononucleate) automated method, 6000 cells per replicate x3 replicates = 18,000 cells. -24 well plate format. -48 hours exposure to loaded PDMS discs. -Media contains serum (10%).

Day 2:

Add disc +

preconditioned medium to cells

Day 4:

Remove disc + change

medium

Day 5:

Fix cell nuclei;

Score for MN using automated Metafer system

Day 1: Seed cells Pre-equil

medium o/n

Cut PDMS discs

1x wash ethyl

acetate

2x wash methanol

Store in ddH2O

Saturated B[a]P

solution

Dilute B[a]P solution; add discs

Store in ddH2O

Sample medium at 0, 24h Sample medium (48h)

Methanol extraction Fluorescence HPLC

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Establishing a PD system for B[a]P induced micronucleus formation

18,000 cells per dose

B[a]P induces Mn in MCL5 cells, confirming metabolism (spiked).

• MCL5 cells can metabolise B[a]P to genotoxic metabolites.

• Mn induction and cytotoxicity (reduced RPD) evident.

• Dose dependent effect.

• TK6 cells (no metabolic activity) showed absolutely no effect (not shown).

Shah U-K, Seager AL, Fowler P, Doak SH, Johnson GE, Scott S, Scott AD, Jenkins GJS (2016). Mutation Research (Genetic Toxicology) 808: 8-19.

Passive dosing of B[a]P in an in vitro MCL5 culture system

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Loading solution (% saturation)

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0 2.51 4.74 10.05 18.21 21.57 µM B[a]P

Serum presence increases B[a]P levels in media Shaking during cellular exposure? 24 hr v 48 hr? CYP450 expression induced by PD?

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B[a]P passive dosing 48 hours

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Conclusions • In order to explore and refine the in vitro exposure to test genotoxic

agents, we should investigate more sophisticated in vitro exposure scenarios.

• Chronic dosing can inform us about mutational hazards related to typical human exposure scenarios.

• Passive dosing linked to genotoxicity as an extension of this approach can be informative in the context of hydrophobic compounds.

• Can be used in IVIVE studies

• These approaches will reduce the need for routine in vivo studies.

Acknowledgements. • Fiona Chapman

• Shareen Doak

• Kate Chapman

• Ed Dudley

• Leanne Stannard

• Kulsoom Shah

• Andy Scott

• David Sanders

• Roger van Egmond

Diolch Thank you

B[a]P passive dosing data.

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B[a]P concentration in medium after 48h

Av. MNMN frequency Av. RPD

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B[a]P concentration in medium after 48h (µM) Av. MNMN frequency Av. RPD

Inserts Free discs

Steel posts

The micronucleus (Mn) test

• The Mn test detects mutagens very sensitively. Most mutagens +ve in Mn test (clastogens and aneugens).

• Automation aids dose response analysis, by providing large data sets (10,000 cells per dose analysed).

• Use these dose responses to select LOEL doses for other endpoints (3-4 doses around the LOEL).

Seager et al., (2014). Recommendations, Evaluation and Validation of a Semi-Automated, Fluorescent-Based Scoring Protocol for Micronucleus Testing in Human Cells. Mutagenesis 29: 155–164.

Preconditioned medium

Well Transwell insert Loaded disc

Surgical steel post attached to plate lid Loaded disc Preconditioned medium MCL-5 cells

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Nominal B[a]P dose (μM)

Spiked B[a]P