University of Hawai‘i Cancer Center

How to do it: in vitro and in vivo assessment of mineralHow to do it: in vitro and in vivo assessment of mineral fibers carcinogenicity

Haining Yang, PhDProfessorProfessor

University of Hawai‘i Cancer Center

Yang H et al, PNAS 2006, PNAS 2010, Clinical Cancer Res 2011

Similar physical and chemical characteristics of erionitefrom North Dakota and Cappadocia, Turkey.

Michele Carbone et al. PNAS 2011;108:13618-13623

©2011 by National Academy of Sciences

Size distribution

Michele Carbone et al. PNAS 2011;108:13618-13623

©2011 by National Academy of Sciences

Size distribution

Larson D Carbone M and Yang H J Toxicol Environ Health B Crit Rev 2016

©2011 by National Academy of Sciences

Larson D,… Carbone M and Yang H. J Toxicol Environ Health B Crit Rev. 2016

In Vitro assays

( ) ( ) S ( )A B C

To check cytotoxicity, cell death

LDH Assay (48 hrs) Alamar Blue (48 hrs) MTS Assay (48 hrs)

To check HMGB1 release, and to check the stimulation of TNF α secretionstimulation of TNF-α secretion

C: Unexposed HMND: North Dakota ErioniteOR O E i itOR: Oregon ErioniteTUR: Cappadocia ErioniteASB: Crocidolite Asbestos

-Yang et al, PNAS, 2006

-Yang et al, PNAS, 2010

-Carbone et al., PNAS 2011

crocidolite asbestos

crocidolite+TNF-α crocidolite+TNF-α

1 week 1 month

(HM/macrophages co-culture system)

Asbestos

Similar biological activity of erionite from North Dakota and Cappadocia, Turkey.

Michele Carbone et al. PNAS 2011;108:13618-13623

©2011 by National Academy of Sciences

Fibrous palygorskite is abundant in th N dsouthern Nevada

Environmental Environmental Environmental Environmental sample #1

Environmental sample #2

Environmental sample #3

o e tasample #4 Purified palygorskite

SampleLength

min/max (µm)Thickness

min/max (µm)

Environmental sample #1 0.2/0.7 0.02/0.06

Environmental sample #2 0.22/1.1 0 04/0 1Environmental sample #2 0.22/1.1 0.04/0.1

Environmental sample #3 0.1/2.6 0.03/0.09

0 4/3 0Environmental sample #4 0.4/3.0 0.01/0.04

Purified Palygorskite 0.16/4.0 0.015/0.04

Palygorskite displayed cytotoxicity towards HM cells and reduced their viability, but roughly half as y, g ymuch as crocidolite

LDH Assay (48 hrs) Alamar Blue (48 hrs)

HM cell + Media HM cells + Crocidolite HM cells + Palygorskite

Crocidolite exposure leads to increases in HMGB1 levels, but palygorskite exposureHMGB1 levels, but palygorskite exposure does not.

Long-term in vivo carcinogenesis study using Balb/c miceusing Balb/c mice

● Balb/c mice were injected● Balb/c mice were injected intraperitoneally twice, one week apart with 1 mg glass palygorskiteapart, with 1 mg glass, palygorskite, or crocidolite.

● Observed for signs of disease for up● Observed for signs of disease for up to one year.

Only mice exposed to crocidolite developed ascites and mesotheliomadeveloped ascites and mesothelioma

Group Ascites MesotheliomaGroup Ascites Mesothelioma

Balb/c - Glass (2x1 mg)* 0/3 (0%) 0/3 (0%)

B lb/ P l kit (2 1 )* 0/19 (0%) 0/19 (0%)Balb/c - Palygorskite (2x1 mg)* 0/19 (0%) 0/19 (0%)

Balb/c - Crocidolite (2x1 mg)* 5/16 (31.3%) 3/16 (18.8%)* In mice exposed to glass or fiber for up to one year.

Crocidolite (H&E)

p g p y

Crocidolite (WT1)

Long-term in vivo carcinogenesis study i M TA iusing MexTAg mice

● MexTAg are transgenic mice that SV40 l T ti iexpress SV40 large T antigen in

mesothelial cellsmuch more susceptible to asbestos-much more susceptible to asbestos-induced carcinogenesis.Good model to interrogate the gcarcinogenic potential of fibers, even fibers with low carcinogenic potential.

● Mice were injected twice one month● Mice were injected twice, one month apart with:

Glass (6 mg) Palygorskite (6 mg)Glass (6 mg), Palygorskite (6 mg), Crocidolite (3 mg).

Only MexTAg mice injected with crocidolite developed ascites andcrocidolite developed ascites and mesothelioma

Group Ascites Mesothelioma Hyperplasia

MexTAg Glass (2x6 mg)p 0/15 (0%) 0/15 (0%) 0/15 (0%)MexTAg - Glass (2x6 mg)p 0/15 (0%) 0/15 (0%) 0/15 (0%)

MexTAg - Palygorskite (2x6 mg)p 0/18 (0%) 0/18 (0%) 0/18 (0%)

MexTAg Crocidolite (2x3 mg)p 14/14 (100%) 13/14 (92 9%) 14/14 (100%)MexTAg - Crocidolite (2x3 mg)p 14/14 (100%) 13/14 (92.9%) 14/14 (100%)

p In mice exposed to glass or fiber for 6 or more months.

Crocidolite (H&E) Crocidolite (WT1) Palygorskite (H&E)

Percentages of macrophages and neutrophils in the peritoneal cavity 3 days after palygorskite exposure p y y p yg pare significantly less than the percentages after crocidolite exposure

Glass Crocidolite42

1Palygorskite

0.3% 47% 11%

Ly6G

Bv4

6% 26% 11%

F4/80 AF488

Inflammation induced by palygorskite was t t i d i fl ti i d dnot as sustained as inflammation induced

by crocidolite

Macrophages remain mostly M1 (anti-tumoral) after continuous palygorskite exposure, whereas M1 macrophages decrease dramatically and M2 (pro-tumoral) macrophages increase after continuous crocidolite exposure

Glass PalygorskiteCrocidolite0 2% 3 8% 10 3% 84 6% 0 6% 23 6%

crocidolite exposure.

D20

6 A

PC

0.2% 3.8% 10.3% 84.6% 0.6% 23.6%

CD86 PE

CD

0.5% 95.7% 4.2% 0.9% 1.3% 74.6%

CD86 PE

Summary and conclusionsSu a y a d co c us o s

● No mice exposed to palygorskite developed mesothelioma.P l ki i d i ifi l l i fl i● Palygorskite induces significantly less acute inflammation.

● Palygorskite causes less sustained inflammation. ● When mice were continuously exposed to palygorskite● When mice were continuously exposed to palygorskite,

they did not develop hyperplasia.● Mice continuously exposed to palygorskite had more M1

macrophages and less M2 macrophages than micemacrophages and less M2 macrophages than mice continuously exposed to crocidolite.

● These specific immune system changes that occur after t i i fib b f l hexposure to carcinogenic fibers may be useful when

interrogating the carcinogenic potential of other mineral fibers.

● Palygorskite is not a likely contributor to mesothelioma development in southern Nevada.

Aloha and Mahalo!

Cases of mesothelioma in southern Nevada that appear to be caused by environmentalthat appear to be caused by environmental exposure to carcinogenic mineral fibers

J Thorac Oncol. 2015 May;10(5):731-7Baumann, Francine; Buck, Brenda J; et al.

Palygorskite backgroundyg g

● Palygorskite is a non-regulated, fibrous, hydrated high-magnesium phyllosilicatehydrated, high-magnesium phyllosilicate mineral.

● International Agency for Research on Cancer● International Agency for Research on Cancer (IARC) to declare that palygorskite fibers > 5 µm are possibly carcinogenic to humansµm are possibly carcinogenic to humans (Group 2B)

● Fibers < 5 µm there is inadequate evidence● Fibers < 5 µm, there is inadequate evidence and carcinogenicity to humans is inconclusive and not classifiable (Group 3)and not classifiable (Group 3)