PlantToxins Duringer2005 by Z1Z6njB

VIEWS: 2 PAGES: 57

									Jennifer M. Duringer, PhD
                    Overview
I.        General introduction
II.       Toxicoses
      •    Tansy ragwort
      •    Tall fescue
      •    Perennial ryegrass
III. Service ~ endophyte toxins
IV. Research
      •    Tall fescue
      •    Perennial ryegrass
                  Plant v. Herbivore
   Plant defenses
       Physical
         thorns, spines, leaf hairs
         plant avoidance by herbivore

       Chemical
         reduce palatability
         adversely affect health of herbivore

               9 classes of alkaloids
               Plant v. Herbivore
   Animal defenses
       Ruminal microbes
           Degradation of plant and fungal toxins
                Plant v. Herbivore
   Animal defenses
       Hepatic metabolism
           Phase I: expose or add functional group
              Cytochrome P450s

              Flavin-containing monooxygenases

              Carboxylesterases

           Phase II: conjugation and elimination
              UDP-glucuornosyltransferases

              Glutathione-S-transferases
                     Toxic Plants
   Toxic plants account for 8.7% of all livestock
    morbidity and mortality
       50% pathogens (bacteria and viruses)
       25% metabolic disorders
       15% parasitic

Toxic plants of interest in the Pacific
             Northwest
   Tansy ragwort
   Tall fescue
   Perennial ryegrass
  Tansy ragwort
(Senecio jacobaea)
    Distribution of Pyrrolizidine
              Alkaloids
   More than 350
    PAs isolated from
    over 6000 plant
    species
       3% flowering
        plants
Toxicity of Pyrrolizidine Alkaloids
    Acute
        rare under natural conditions
        liver necrosis  death
    Chronic                                Normal hepatocytes
        veno-occlusive disease
             cirrhosis
        lungs, heart
    Cancer
        bind macromolecules                Hepatocytes with
                                          veno-occlusive disease
             DNA: interrupt cell cycle
         Livestock Exposure to
         Pyrrolizidine Alkaloids
   Food supply
       Senecio and Crotalaria genuses
            Senecio jacobaea
   Poisoning
       livestock losses
            $20 million/year in Pacific Northwest
            10% cattle, 5% small stock deaths/year due
             to plant poisoning in South Africa
    Differences in Susceptibility
           to PA Toxicity
   Age
       young > adult
   Gender
       depends on species
   Species
    Differences in Susceptibility
           to PA Toxicity
   Metabolism by ruminal microbes
       Bacteria, fungi, protozoa




   Intestinal absorption
   Hepatic metabolism
      Major PAs in Tansy Ragwort

                HO CH3                    HO CH3                       HO CH3
          O
                         O                           O                          O
H3C                          H3C                         H3C
                CH3 O                    CH3 O                        CH2 O
      O       O H                  O   O H                     O    O H


                N                          N                            N

              Jacobine                 Senecionine                 Seneciphylline
PA-Degrading Enrichment L4M2
                              140                               0.09
                              120                               0.08
   PA Concentration (ug/ml)




                                                                                          MPN PA Deg. Microbes/ml
                              100                               0.07                107
                               80                               0.06




                                                                       600nm O.D.
                                                                0.05
                               60
                                                                0.04
                               40
                                                                0.03
                               20                               0.02
                                0                               0.01                106
                                    0   2      4        6   8
                                            Time (hr)



Resistance from sheep can be transferred to cattle
Protect animals with a
     “Probiotic”
    L4M2 Identification ~
   Culture-Based Methods


Coccobacillus   Gram (–)
Curved Rod      Gram (–)
Long Rod        Gram (–)
Small Coccus    Gram (+)
       L4M2 Characterization
   Individual members of L4M2 were not able
    to degrade PA

   5-10% of ruminal bacteria are culturable

How do you identify viable “not yet
   cultured” ruminal bacteria?
               Genetic Approach
   Find molecule or gene common to all
    organisms

   Molecule must have been
    structurally/functionally present over a long
    period of time

   Molecule must contain enough variability to
    distinguish between species of organisms
       16S rRNA
           LH-PCR
           RFLP
        Results ~ LH-PCR
Peak      Length
 1        342 bp
 2        345 bp
 3        349 bp                      7
 4        355 bp            2 4

                            13
 5        358 bp
 6        365 bp            1
                                 5
 7        369 bp                  6

 Seven possible bacterial
 species present in L4M2
            Results ~ RFLP
   Lane 1 – Ladder            1 2 3 4 5 6 7 8
   Lane 2 – 119
   Lane 3 – 115
   Lane 4 – 121
   Lane 5 – 101
   Lane 6 – 146
   Lane 7 – 124
   Lane 8 - 142

     Six RFLP patterns = six possible bacterial species
     present in L4M2?
Identification of L4M2 Members
        Sequence RFLP and LH-PCR Clones


        Evaluate Sequences
        Genbank and RDP

                                          1
1:   5’-GGCCGGCTATGGC-3’                  2
2:   5’-GGCCGGCTATGGC-3’                  4
3:   5’-GGCCGGCAATGGT-3’
4:   5’-GGCCGGCAATGGC-3’                  3
                             Phylogenetic tree
Subgroup:
Desulfovibrio
desulfuricans


Group:
Bacteriodes


Group:
An. thermoterrenum
Subgroup:
Clostridium leptum

Group:
Clostridium coccoides

Group:
Sporomusa

Subgroup:
Veillonella parvula
        Summary of Molecular
           Experiments
   Synergistic relationship between members
    of L4M2
   LH-PCR indicated 7 individual organisms in
    L4M2
   RFLP analysis showed that 2 of the 7 had
    the same restriction map
   Phylogenetic analysis grouped the bacteria
    into 6 groups
 Compare hepatic in vitro
   metabolism of the
  pyrrolizidine alkaloid
senecionine in sheep and
          cattle
Hepatic Metabolism of Senecionine




             TOXIC

                     NON-TOXIC
 Generally
NON-TOXIC
In vitro incubations with senecionine
                                       Senecionine
              NADPH                      N-oxide
                             DHP
                                                                        Senecionine
                 D




                           DHP           SN N-oxide
                         (nmol/min/       (nmol/min/       Ratio            SNA/SNB
    Species     Gender   mg protein)      mg protein)   (DHP/N-oxide)         (%)
     Cattle     Female   0.42±0.13       0.33±0.08       1.24±0.19          88.6±2.1
     Cattle      Male    0.39±0.08       0.19±0.04       2.11±0.18          87.0±2.0
    Cattle       Both    0.41±0.01       0.26±0.07       1.68±0.29          87.8±1.9
    Sheep       Female   0.28±0.09       1.11±0.38       0.29±0.05          76.4±2.1
    Sheep        Male    0.50±0.10       1.08±0.06       0.46±0.09          73.5±4.6
    Sheep        Both    0.39±0.11       1.10±0.26       0.37±0.08          74.9±3.4
      Chemical Inhibition of DHP and
           N-oxide formation
                                                     Chemical Inhibitor


                               10 µM          0.25 mM             0.25 mM              1.0 mM
             0.5 mM          Triacetyl-      Methimazole          Thiourea         Phenylmethyl-        0.1 mM Tri-o-
            SKF-525A       oleandomycin        (pH 8.5)           (pH 8.5)        sulfonyl fluoride   cresyl phosphate

           DHP     NO       DHP     NO       DHP      NO        DHP       NO        DHP      NO         DHP     NO

Species   (as % control)   (as % control)    (as % control)     (as % control)     (as % control)      (as % control)


Female    15.3±    49.2±   61.4±    67.4±   35.3±     16.4±    35.4±      26.0±   87.3±      54.2±    23.8±     46.5±
Sheep      3.1*    10.6*    2.9*    14.3*   11.7*      2.7*    10.2*       6.6*    4.1       13.7*     2.9*      9.8*



 Male     12.7±    56.3±   50.9±    83.6±   25.3±      4.8±    25.4±      19.6±   90.2±      51.9±    19.2±     48.8±
Sheep      4.1*     3.3*    2.2*     2.2*    6.4*      0.3*     3.7*       3.4*    4.2        7.3*     2.9*      3.6*


Female    22.1±    48.7±   17.6±    55.9±   23.9±     14.1±    59.1±      24.2±   116.3±     84.1±    25.1±     47.6±
 Cattle   10.6*     7.9*    0.7*    11.3*    8.3*      4.4*    15.8*       5.3*     9.1      19.1      2.2*      7.8*


 Male     12.6±    35.9±   18.0±    52.0±   39.1±     22.8±    71.6±      34.4±   113.7±     84.7±    21.4±     51.3±
 Cattle    2.9*     5.2*    2.7*     9.7*    8.8*      4.3*     8.9*       4.3*     8.5      10.9      2.4*      3.1*
                                                         CYP3A and CYP2B Activity
Testosterone hydroxylation (nmol/min*nmol P450)




                                                   0.3   6 beta-hydroxytestosterone
                                                         16 beta-hydroxytestosterone                            No differences in
                                                  0.25
                                                                                                                 formation between
                                                   0.2
                                                                                                                 species or genders
                                                  0.15


                                                   0.1


                                                  0.05


                                                    0
                                                         Female      Male cattle       Female   Male sheep
                                                          cattle                        sheep
                                                                   FMO Activity
                               9.0

                               8.0                                                                     Sheep > cattle by 2X
                               7.0

                                                                                                        No gender differences
TNB-oxidation/min/mg protein




                                                                                                    
nmol methimazole-enhanced




                               6.0

                               5.0
                                                                                                        for either species
                               4.0

                               3.0

                               2.0

                               1.0

                               0.0
                                     Cattle   Sheep   Female    Male Cattle   Female   Male Sheep
                                                       Cattle                 Sheep
          Summary of Metabolic
             Experiments
   Differences
       N-oxide formation and FMO activity (sheep >
        cattle)
       amount of parent compound metabolized
        (sheep > cattle)
       role of CYP3A (cattle > sheep)

   Similarities
       DHP formation
       role of P450s, FMOs, esterases by chemical
        inhibition
        Conclusion

 Increased ruminal metabolism in
sheep appears to be an important
protective mechanism with hepatic
 enzymes providing a secondary
 means to degrade PAs which are
     absorbed from the rumen
      Tall fescue
(Festuca arundinacea)
Tall fescue (Festuca arundinacea)
   Perennial cool-season grass
   35 million acres in the US
       Over 70% of the world’s supply grown in Pacific
        NW → 2nd largest commodity in OR (~$400
        million/year)
   Livestock feed, turf grass, erosion control
   Infected with Neotyphodium coenophialum
       Endophyte
            good for the plant: stress tolerance & drought resistance
            bad for livestock: produces ergot alkaloids
                 responsible for insect and mammalian toxicoses
        Tall fescue
            Ergovaline
              H CH3 OH
        O     N   O
                                   N
        H                      H
                           N
                  O                    O
              N            H       CH(CH3)2
                  CH3
              H
              Ergovaline

    N
H
    Etiology of Fescue Toxicosis
   α-adrenergic agonists
       stimulate smooth muscle cell contraction →
        vasoconstriction
       necrosis and sloughing of
        extremities
       ↓ed weight gain (ADG)
   dopamine agonists
       inhibit production of prolactin
       ↓ed milk production and reproductive
        efficiency
               Clinical Diseases
   Summer syndrome or summer slump
       heat intolerance
       d grazing = d weight gain
       reproductive difficulties
   Fescue foot
       cold intolerance
       necrosis  gangrene  sloughing
   Fat necrosis
       disrupts digestion and calving
Threshold Levels for Clinical
         Disease
          Species                  Ergovaline (ppb)
          Horses*                      300-500
           Cattle                      400-750
           Sheep                       500-800

 *Except for mares in the last 60 to 90 days of pregnancy,
 when the threshold is zero.
       The Consequences
Decrease in production of $1 billion/year
       for the livestock industry
        Ruminal Metabolism of Ergot
                 Alkaloids                            O
                                                          H
                                                          N
                                                            CH3 OH
                                                              O
                                                                    N
                                                                  H
   Bioavailability                                             N
                                                                              O
                                                              O
       relatively unknown                                N     H3C
                                                              CH3
                                                                            CH3
                                                          H
       conversion to lysergic acid? other                    Ergovaline
        compounds?                            N
                                              H
   Transport across tissue wall
       rumen > omasum > reticulum           Ergonovine           H
       ergoline > ergopeptides                           O       N
                                                                             OH
                                                          H
   Rumen fluid                                                       CH3


       degradation of ergovaline, other                          N
                                                                      CH3
                                                                  H
        ergot alkaloids
                                                      N
                                                  H
        Hepatic Metabolism of Ergot
                 Alkaloids                   O
                                                    H CH3 OH
                                                    N   O
                                                                         N
                                             H                      H
                                                                N
                                                        O                    O
   Cytochrome P450s                                N
                                                          CH3
                                                                H       CH2Ph
                                                    H
       rats, humans: CYP3A                           Ergotamine
                                                      Ergotamine
       livestock: CYP3A?             H
                                        N
                                                  H CH3 OH
       hydroxylation on proline ring        O    N   O
                                                                        N
                                             H                      H
       excrete in bile within 6 hours                          N
                                                                             O
                                                      O
                                                  N             H       CH(CH3)2
                                                        CH3
                                                  H
                                                   Ergovalinine
                                                 Ergocristine
                                         N
                                     H
    Research Strategy ~ Microbes
           That Degrade
   In vivo studies
       Steer and sheep studies
            4X4 Latin square
            Varying levels of ergovaline supplied by E+ tall
             fescue straw
            Samples collected
                 Ruminal fluid, serum, urine, feces
                    Ergovaline, lysergic acid, lolines, digestiblity of straw,

                     VFA, Ruminal NH4
                    Rumen fluid: isolation of ruminal bacteria that degrade
                     ergovaline and/or lysergic acid → development of a
                     probiotic
    Research Strategy ~ Microbes
           That Degrade
   Changes in microbial ecology to eludicate
    adaptation mechanism
       Real-time PCR
       Whole ruminal fluid and enrichments of ruminal
        bacteria from steers or sheep on E+ straw
 Research strategy ~ Hepatic
Metabolism of Ergot Alkaloids
1. Determine whether the genetic divergence
   remained after 13 generations of relaxed
   selection
2. Characterize the metabolism of ergotamine in
   mouse liver microsomes from R and S lines
   before and after exposure to E+ feed
3. Identify the major microsomal
   transformation products from metabolism
   of ergotamine by LC/MS/MS
                Research strategy ~ Hepatic
               Metabolism of Ergot Alkaloids
     200




     180




     160




     140

                                      Metabolite 1
     120
mV




                                                                          Metabolite 1 epimer
     100


                                    Metabolite 2
      80

                                     Ergotamine
      60
                     Metabolite 5                                     Metabolite 2 epimer
                     Metabolite 4                                            Ergotamine epimer
      40

                          Metabolite 3
      20




           0     5       10         15        20      Time (minutes) 35
                                                     25      30                  40       45     50   55
          LC/MS: Ergotamine and ergotamine
                       epimer
Ergot #109-123 RT: 1.11-1.25 AV: 15 NL: 2.26E8
T: + c Full ms [ 100.00-800.00]
                                                                                                                                     582.3
              100
                                                                                    9'
                      95                                             HO
                                                          H3C                            8'
                      90                                         O
                                           O             NH                     N
                      85                                                    H
                                                                      N
                      80                                                                 O
                      75                                        O
                      70                                 N
                                                               CH3
                      65                             H
 Relative Abundance




                      60

                      55
                                   HN
                      50

                      45

                      40
                                                                                                                                         583.3
                      35

                      30

                      25

                      20
                                                                                                                                 564.3
                      15

                      10                                                                                                                  604.2
                       5                         223.3
                                         208.3                                                                                                 605.2
                           121.1 149.1                   242.4       301.2 320.2         366.5     415.6 430.9   507.6 522.8                                 683.7 702.6   740.0 771.5 786.5
                       0
                        100       150     200            250          300           350          400       450     500         550       600           650         700       750        800
                                                                                                           m/z
                                       LC-MS/MS: Metabolites 1,2 and
                                              Their Epimers
Metabolite2 #322-352 RT: 6.52-7.08 AV: 31 NL: 2.36E5
T: + c Full ms2 598.30@40.00 [ 160.00-600.00]                                                           OH
                                                                                                                                                597.5
              100                                                                             9'
                      95                                                      HO
                                                             H3C                                   8'
                      90
                                               O            NH
                                                                        O
                                                                                          N
                                                                                                          OH
                      85                                                              H
                                                                                N
                      80
                                                                                                   O
                      75                                                O
                      70
                                                            N
                      65                                            CH3
                                                            H
 Relative Abundance




                      60

                      55

                      50      HN
                      45

                      40

                      35

                      30
                                                                   297.2
                      25                                                                                                                             598.5
                      20                                  268.1
                      15                                                    320.0
                                            223.2                                                                                     562.1 580.1
                      10
                                                                277.0
                       5               208.2        265.9                                                                      519.1 552.1
                              191.2                                            323.1 341.2 379.4         439.2 467.2 506.6                            599.2
                       0
                        150           200           250             300             350        400           450         500          550           600       650   700   750   800
                                                                                                                   m/z
                       LC-MS/MS: Metabolites 3,4 and 5
                                                                                                OH
Metabolite5 #96-132 RT: 3.07-4.47 AV: 37 NL: 8.82E3
T: + c Full ms2 614.30@38.00 [ 165.00-700.00]
                                                                                      9'                                                           596.1
                  100                                                   HO
                                                          H3C                              8'
                      95                                            O                            OH
                                            O            NH                       N
                      90                                                      H
                                                                          N                                                                            614.4
                      85
                                                                                           O
                      80                                        O
                      75                                 N
                      70                                      CH3
                                                        H
                      65
 Relative Abundance




                      60
                      55      HN
                      50
                      45
                      40
                      35
                      30
                      25
                      20                                                  320.0
                      15                 223.2              276.9
                                                 268.1
                      10
                                                                292.1
                      5             221.2                                                                          467.2                   568.2           615.5
                           191.2                 249.1
                                                                    310.1 329.0 348.0            407.5 420.2               485.2   553.1                           646.0 663.1
                      0
                                   200            250               300           350                400         450         500     550            600            650           700
                                                                                                           m/z
Perennial ryegrass
 (Lolium perenne)
               Perennial Ryegrass
   Perennial cool-season bunchgrass
   ~250,000 acres in the US
   High palatability and digestibility
       Ideal for livestock
   High yield and fast establishment
       Ideal in golf courses and sports fields
   Infected with Neotyphodium lolii
       Endophyte, produces loline alkaloids
            good for the plant: deters insect pests
            bad for livestock: responsible for staggers
        Perennial ryegrass
                Lolitrem B
            CH3
        O    CH3
H 3C
 H 3C
                                OH       H 3C      CH3
        O
                                 O
                                         O
                   N H C                           H
                        3
                   H     H 3C                O
                                     O
              Lolitrem B
                    .                        CH3
                                     H 3C
    Etiology of Ryegrass Staggers
   Inhibit the inhibitory GABA neurotransmitter
       Leads to dysinhibition or excitement, seen as
        tremors or staggering in livestock
   Inhibit calcium-activated potassium
    channels
       Leads to membrane depolarization →
        vasocontriction → increase in blood pressure
        and contractile activity of the gut
   All species are affected
             Clinical Disease
   Gross incoordination when disturbed
   Limbs not flexed
   Fall easily, struggling violently when attempting
    to rise.
   Outbreaks in late summer when pasture is short.
    Disappears when rain stimulates pasture
    growth.
Threshold Levels for Clinical
         Disease
          Species                    Lolitrem B
                                        (ppb)
          Horses*                  Not determined
           Cattle                    1800-2000
          Sheep                      1800-2000
 *Except for mares in the last 60 to 90 days of pregnancy,
  when the threshold is zero.
            The Consequences
   “An inconvenient disease”
       Causes almost no fatalities
       Removal from feed alleviates the problem
Ruminal and Hepatic Metabolism
      of Loline Alkaloids




             ???
    Research Strategy ~ Metabolism
          of Loline Alkaloids
   Determine whether lolitrem B appears in urine
    and feces of cattle fed high lolitrem B ryegrass
       As itself                           O
                                              CH3
                                               CH3
                                 H 3C
       As a conjugate            H 3C
                                                                                H 3C        CH3
       As a metabolite                  O                        OH
                                                                   O
                                                                                O
                                                     N H C                                  H
   Resting place in the fat?                        H
                                                          3
                                                           H 3C
                                                                       O
                                                                                    O
                                                Lolitrem B                          CH3
       Miyazaki 2004 paper                   CH3
                                                                       H 3C

       Rumen = neutral pH,     H 3C
                                         O     CH3

        abomasum = acidic pH     H 3C
                                        O                         OH
            Lolitrem B vs E                                       O
                                                                                    OH
                                                     N H C
                                                        3
                                                     H    H 3C                          O
                                                                           O
                                                Lolitrem E                              CH3
                                                                               H 3C

								
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