MERCURY and THE IMMUNE SYSTEM: OLD WINE IN NEW BOTTLES?
ELLEN SILBERGELD
JOHNS HOPKINS SCHOOL OF PUBLIC HEALTH DEPARTMENT OF ENVIRON HEALTH SCIENCES SPRING 2005 MEETING AE-SOT PITTSBURGH
ACKNOWLEDGEMENTS
• STUDENTS and COLLEAGUES – Prof Donna Mergler, UQAM – Dr Jennifer Sass, NRDC – Dr Peter Crompton, Harvard – Ines Silva, Univ of Michigan – Dr Jennifer Nyland, Dr Noel Rose, Dr DeLisa Fairweather, Johns Hopkins – Dr Charles Via, Dr John Sacci, Dr G T Strickland, Univ Maryland – Dr Emma Calderon Aranda, CINVESTAV – Mexico – Dr Jose Maria de Souza, Dr Ana Maria Ventura, Dr Elisabeth Santos, Ms Iracina de Jesus FNS-IEC -Brazil
FUNDING
• NATIONAL INSTITUTES OF HEALTH • PAN AMERICAN HEALTH ORGANIZATION • ARTHRITIS FOUNDATION • AMERICAN HEART ASSOCIATION
• CURE AUTISM NOW FOUNDATION
MERCURY IN THE ENVIRONMENT
• INCREASINGLY RECOGNIZED AS A GLOBAL POLLUTANT (UNEP, WHO) • MAJOR ENVIRONMENTAL RISK TO CHILDREN’S HEALTH (EPA, WHO, CEC) • CRITICAL EFFECT - DEVELOPMENTAL NEUROTOXICITY (WHO, NAS) • FISH CONSUMPTION MAJOR ROUTE OF HUMAN EXPOSURE TO MeHg • AIRBORNE Hg EXPOSURES? • MERCURY EXPOSURES CONTINUE IN WORKPLACES
THE CONTINUING PROBLEM OF METHYL MERCURY IN FISH: HOW MUCH METHYLMERCURY IS THERE IN TUNA?
• 0.5-1.0 ppm CONSIDERED “SAFE” (EPA/FDA) • 2-10 ppm FISH CONSUMPTION CAUSES PROBLEMS • 10 ppm = 10 ug/gm; • In 6 oz ≈ 1.8 mg • FOR A 70 KG PERSON, DOSE = 26 ug/kg
MERCURY RISKS and CAUGHT FISH CONSUMPTION
MERCURY HAZARDS are LOCAL, REGIONAL and GLOBAL
TOXIC EFFECTS OF MERCURY COMPOUNDS
• NEUROTOXICITY
– DEVELOPMENTAL EFFECTS (NRC, WHO) – ADULTS MAY BE AS SENSITIVE
• NEPHROTOXICITY • DERMATOTOXICITY • IMMUNOTOXICITY?
HOW ARE IMMUNOTOXIC MECHANISMS INVOLVED in MERCURY TOXICITY
• NEPHROPATHY – IMMUNE COMPLEX (GBMP Abs)
• NEUROTOXICITY – INHIBITION OF NEURAL MIGRATION [DEV, NEURODEGEN DISEASE] • IMMUNE SUPPRESSION • AUTOIMMUNE DYSFUNCTION
IMMUNOLOGIC MECHANISMS ARE INVOLVED IN DEVELOPMENTAL NEUROTOXICITY OF MERCURY (Sass et al 2001; Calderon et al in press)
• Mercury induces glial activation • Microglia are macrophage lineage cells • Microglia direct neuronal migration through cytokine/chemokine/CAM pathways Developmental mercury exposure affects these signaling pathways – signal transduction events in cerebellar cell cultures from neonatal mice – altered gene expression, cytokine production, and cellular protein levels
AUTOIMMUNE DISEASE
• COMPLEX DISORDERS (ORGAN SPECIFIC and SYSTEMIC) • BASIC MECHANISM: FAILURE TO RECOGNIZE “SELF” • RISK FACTORS INCLUDE GENETICS and ACQUIRED EXPOSURES • STRONG SEX BIAS IN DISEASE INCIDENCE AND SEVERITY • GEOGRAPHIC PATTERNS OF DISEASE • POORLY MANAGED, INCURABLE DISEASES
AUTOIMMUNE DISEASE – A WOMEN’S HEALTH ISSUE
MERCURY AND AUTOIMMUNE DISEASE
• CAN MERCURY CAUSE AI DISEASE? – HUMAN DATA? • NEPHROTOXICITY MAY INVOLVE AUTOIMMUNE MECHANISMS • FRANK AI DISEASE NOT DEMONSTRATED IN OCC STUDIES BUT EPI DATA ARE LIMITED • NEUROTOXICITY MORE SENSITIVE OUTCOME – EXPERIMENTAL DATA – YES • INBRED RODENT STRAINS ARE SUSCEPTIBLE • RESPONSE CAN INCLUDE AUTOANTIBODIES, VASCULITIS, NEPHROPATHY • DOSES ARE RELATIVELY HIGH
MERCURY and HUMAN AUTOIMMUNE DISEASE: THE LATEST STUDY!
• CAROLINA LUPUS STUDY • CASE:CONTROL (265:355) • CASES: 90% FEMALE, 60% AFRICAN AMERICAN • INCREASED ODDS FOUND FOR:
– OCCUPATIONAL EXPOSURES TO MERCURY
– EMPLOYMENT AS DENTAL TECHNICIAN – MIXING PESTICIDES (NOT IDENTIFIED) Cooper et al J Rheumatol 2004: 31: 1928-1933
CAN MERCURY ACCELERATE AI DISEASE? [the BASF approach]*
EXPERIMENTAL DATA
– Hg accelerates pathology in lupus-prone strains of mice (NZB, BXB) – Hg accelerates disease in Graft Versus Host model of LUPUS, Cardiac Myosin model of autoimmune myocarditis and cardiomyopathy (Via et al 2003; Nyland et al 2003, 2004)
*“WE DON’T CAUSE DISEASE; WE JUST MAKE IT WORSE”
Hg and GVHD/LUPUS
• The model: C57Bl/6 x DBA/2 – F1 • DBA/2 females + B6D2-F1 pretreated with iHg, 20 or 200 mcg/kg a.d. for 15 da. • Transfer maternal splenocytes (100 cells) • Chronic SLE-like disease develops over 18-20 months • Signs include: proteinuria, tubular nephropathy, vascular damage, autoABs [anti-ss-DNA; ANA]
GVHD/SLE: iHg accelerates death
Hg ACCELERATES AUTOIMMUNE NEPHROPATHY
PROTEINURIA IN GVHD+/-Hg
2.5 Control Mercury Tx
Mean Proteinuria Score
2
1.5
1
0.5
0
1
2
3
4
Time since graft (months)
Hg increases serum antinuclear antibodies - GVHD +/- Hg
A B
C
D
Hg increases serum anti-SS DNA antibodies
Hg and Autoimmune Myocarditis
• AM leading cause of sudden cardiac failure in young; post-infection autoimmune disease • BALB/c female mice pretreated with iHg 10, 20, 100 or 200 μg/kg a.d. for 15 da • EAM induced by injecting antigen [cardiac myosin peptide (CMP)] + CFA + pertussis toxin or by infection with Coxsackie B3 virus • CMP – by 30 da, cardiomyopathy develops characterized by cardiac enlargement, arrhythmias, IgG1/2 [antiCMP ABs]; • CB3V – after infection and viral clearance, CM develops by 35 da
Murine model of myocarditis used
• CB3 virus-induced model (BALB/c) – not susceptible to iHg-induced autoimmune disease
40 35
Myocarditis (% inflammation)
30 25 20 15 10 5 0 3
Acute phase
BALB/c
Chronic phase
7
10
14
18 21 28 Days pi
35
42
56
CARDIOMYOPATHY +/- MERCURY: Hg exposure prior to antigen
A/J male mice
PREVALENCE
50%
77%
92%
0
EAM alone
EAM + iHg10
EAM + iHg100
iHg100 alone
iHg treatment prior to CB3V infection increases myocarditis
A. Myocarditis Day 12pi Pretreatment
Myocarditis (% inflammation)
30 DC: 50% 25 DC: 60% 20 15 10 5 0 PBS Mercury
B. Myocarditis Day 35pi Pretreatment
Myocarditis (% inflammation)
50 DC: 100% 40 30 DC: 30% 20 10 0 PBS Mercury
**
*
HISTOPATHOLOGY OF EXPERIMENTAL AUTOIMMUNE MYOCARDITIS:
Control + CB3V Hg + CB3V
CAN WE FIND BIOMARKERS OF HgINDUCED AUTOIMMUNITY IN HUMANS?
• INCONSISTENT FINDINGS IN WORKERS, BUT ONLY STUDIES OF MEN EXPOSED TO iHG • NO FRANK AUTOIMMUNE DISEASE ASSOCIATIONS • RELATIVELY NONSPECIFIC MARKERS USED – IMMUNOGLOBULINS, CELL SUBSETS
OUR STUDY: Hg exposures and autoimmunity in gold workers
• POPULATION – ARTISANAL GOLD MINERS and RIVERINE POPULATIONS • LOCATION – AMAZONIAN BRAZIL • CENSUS BASED SAMPLE and CONVENIENCE SAMPLE (98; 132) • Hg EXPOSURE BIOMARKERS (HAIR and URINE) • HEALTH/OCCUP-RES-DIET INFO by QUESTIONNAIRE, CLINICAL EXAM • SERUM COLLECTIONS
Small (Artisanal) scale mining: major exposures to elemental, inorganic, and methyl mercury • • • • • • World wide activity – 2 to 6 million persons Women and children Hazardous conditions Toxic chemicals Illegal, unregulated Regional, national, transboundary impacts
AIRBORNE MERCURY LEVELS in GARIMPOS in LATIN AMERICA –WHO guidance <0.01 mg/m3
• Levels near amalgam burning in garimpos >100 • Levels in camps - 0.03-10 • Levels near gold shops in towns >20
ARTISANAL GOLD MINING: AMAZONIA TAPAJÓS WATERSHED, PARÁ BRAZIL
DEFORESTATION: Rio Rato, Tapajos
HYDRAULIC MINING - garimpo RIO RATO, TAPAJOS
AMALGAMATION WITH MERCURY
BURNING THE AMALGAM AT THE GARIMPO
CHILD LABOR IN ARTISANAL MINING
Hg EXPOSURES IN GOLD MINERS – BRAZIL BLOOD Hg LEVELS
AUTOANTIBODIES (ANA, ANoA) IN PERSONS EXPOSED TO INORGANIC Hg
1% 24% Rio-Rato ANA Percentages C) 0 1 10 47% 1 20 1 40 1 80 17% 8% 2% 1% 1 160 1 320
c
1%
4%
Tabatinga-Adults ANA Percentages 2% 0 1 10 1 20 1 40 93%
1% 26%
Rio-Rato ANoA Percentages 0 1 10 1 20
2%
0%
Tabatinga-Adults ANoA Percentages 0% 0 1 10 1 20 1 40
7% 3% 1% 3% 59%
1 40 1 80
E) 160 1
1 320
F)
98%
IS ANTIFIBRILLARIN A BIOMARKER OF Hg AUTOIMMUNITY in HUMANS?
CONCLUSIONS
• Hg is immunotoxic, affecting host response to infections, susceptibility to autoimmune disease, and acting on immune mechanisms in target organ disease • The immunotoxic effects of Hg in animals are the lowest dose/effects yet described (0.4 μg/kg)… • There may be genetic susceptibility factors for Hg immunotoxicity • Hg may play a contributing role in the incidence and severity of autoimmune disease • DO WE NEED TO RE-ASSESS THE RISKS OF MERCURY, ESP FOR ADULTS?
A CONCEPTUAL MODEL FOR Hg AND AUTOIMMUNITY (Step 1)
A CONCEPTUAL MODEL FOR Hg AND AUTOIMMUNITY (Step 2)
A CONCEPTUAL MODEL FOR Hg AND AUTOIMMUNITY (Step 1 + 2 = 3)
IF MERCURY IS ASSOCIATED WITH AUTOIMMUNE DISEASE…
• WHAT ARE THE CRITICAL EXPOSURE BIOMARKERS – CHRONIC, CURRENT? – INORGANIC AS WELL AS ORGANIC? – LATENCIES BETWEEN EXPOSURE/OUTCOME? • DOES HG AFFECT THE DEVELOPING IMMUNE SYSTEM – YES…IN MICE • HOW WOULD WE STUDY THIS? – AUTOIMMUNE DISEASES ARE RARE – CASE:CONTROL? ENRICHED POPULATIONS? • ARE THERE SUSCEPTIBLE SUBPOPULATIONS? – PERSONS FROM AI DISEASE FAMILIES – PERSONS WITH SPECIFIC GENETIC POLYMORPHISMS – WOMEN
PRENATAL MERCURY EXPOSURE AFFECTS IMMUNODEVELOPMENT [Silva et al 2005]
Note: inorganic Hg was used; some similar effects observed with MeHg
MERCURY and ASD
• Do mercury compounds CAUSE or CONTRIBUTE to ASD? – Epidemiological studies are needed • Are the mechanisms of mercury toxicity relevant as MODELS (not necessarily as CAUSES) of the mechanistic processes in ASD? – Examples: APO E -/- mouse and Alzheimers;
6-OHDA and Parkinsonism